CN117903276A - Polypeptide VKTP-Vf13, pharmaceutical composition and use thereof - Google Patents
Polypeptide VKTP-Vf13, pharmaceutical composition and use thereof Download PDFInfo
- Publication number
- CN117903276A CN117903276A CN202311742628.4A CN202311742628A CN117903276A CN 117903276 A CN117903276 A CN 117903276A CN 202311742628 A CN202311742628 A CN 202311742628A CN 117903276 A CN117903276 A CN 117903276A
- Authority
- CN
- China
- Prior art keywords
- polypeptide
- vktp
- amino acid
- seq
- nucleic acid
- 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
- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 168
- 229920001184 polypeptide Polymers 0.000 title claims abstract description 155
- 102000004196 processed proteins & peptides Human genes 0.000 title claims abstract description 155
- 239000008194 pharmaceutical composition Substances 0.000 title claims abstract description 18
- 208000007536 Thrombosis Diseases 0.000 claims abstract description 30
- 239000003814 drug Substances 0.000 claims abstract description 22
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract description 19
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims abstract description 14
- 125000000539 amino acid group Chemical group 0.000 claims abstract description 6
- 238000002360 preparation method Methods 0.000 claims abstract description 4
- 108090000790 Enzymes Proteins 0.000 claims description 29
- 102000004190 Enzymes Human genes 0.000 claims description 29
- 238000000034 method Methods 0.000 claims description 21
- 150000003839 salts Chemical class 0.000 claims description 21
- 102000039446 nucleic acids Human genes 0.000 claims description 20
- 108020004707 nucleic acids Proteins 0.000 claims description 20
- 150000007523 nucleic acids Chemical class 0.000 claims description 20
- 239000013598 vector Substances 0.000 claims description 18
- 238000003259 recombinant expression Methods 0.000 claims description 16
- 201000010099 disease Diseases 0.000 claims description 12
- 239000013604 expression vector Substances 0.000 claims description 10
- 238000004519 manufacturing process Methods 0.000 claims description 9
- 239000002773 nucleotide Substances 0.000 claims description 9
- 125000003729 nucleotide group Chemical group 0.000 claims description 9
- 108091026890 Coding region Proteins 0.000 claims description 8
- 230000023555 blood coagulation Effects 0.000 claims description 6
- 239000003112 inhibitor Substances 0.000 claims description 5
- 238000011282 treatment Methods 0.000 claims description 5
- 238000012258 culturing Methods 0.000 claims description 3
- 239000002671 adjuvant Substances 0.000 claims description 2
- 238000003776 cleavage reaction Methods 0.000 claims description 2
- 230000007017 scission Effects 0.000 claims description 2
- 230000000694 effects Effects 0.000 abstract description 50
- 229940079593 drug Drugs 0.000 abstract description 12
- 230000010100 anticoagulation Effects 0.000 abstract description 10
- 230000005764 inhibitory process Effects 0.000 abstract description 8
- 208000032843 Hemorrhage Diseases 0.000 abstract description 7
- 208000034158 bleeding Diseases 0.000 abstract description 7
- 230000000740 bleeding effect Effects 0.000 abstract description 7
- 241000239290 Araneae Species 0.000 abstract description 5
- 241000283070 Equus zebra Species 0.000 abstract description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 abstract description 4
- 230000009465 prokaryotic expression Effects 0.000 abstract description 3
- 238000012827 research and development Methods 0.000 abstract description 2
- 229940088598 enzyme Drugs 0.000 description 31
- 210000004027 cell Anatomy 0.000 description 26
- 108091033319 polynucleotide Proteins 0.000 description 25
- 102000040430 polynucleotide Human genes 0.000 description 25
- 239000002157 polynucleotide Substances 0.000 description 25
- 239000000203 mixture Substances 0.000 description 19
- 238000002474 experimental method Methods 0.000 description 14
- 230000014509 gene expression Effects 0.000 description 14
- 108090000623 proteins and genes Proteins 0.000 description 13
- 239000000523 sample Substances 0.000 description 13
- 108010094028 Prothrombin Proteins 0.000 description 10
- 102100027378 Prothrombin Human genes 0.000 description 10
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 10
- 239000000872 buffer Substances 0.000 description 10
- 229940039716 prothrombin Drugs 0.000 description 10
- 239000007788 liquid Substances 0.000 description 9
- 238000011160 research Methods 0.000 description 9
- 108010000499 Thromboplastin Proteins 0.000 description 8
- 102000002262 Thromboplastin Human genes 0.000 description 8
- 238000009472 formulation Methods 0.000 description 8
- 239000000546 pharmaceutical excipient Substances 0.000 description 8
- 102000004169 proteins and genes Human genes 0.000 description 8
- PXHVJJICTQNCMI-UHFFFAOYSA-N Nickel Chemical compound [Ni] PXHVJJICTQNCMI-UHFFFAOYSA-N 0.000 description 7
- 238000002835 absorbance Methods 0.000 description 7
- 150000001875 compounds Chemical class 0.000 description 7
- 230000036961 partial effect Effects 0.000 description 7
- 239000000243 solution Substances 0.000 description 7
- PGOHTUIFYSHAQG-LJSDBVFPSA-N (2S)-6-amino-2-[[(2S)-5-amino-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-4-amino-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-5-amino-2-[[(2S)-5-amino-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S,3R)-2-[[(2S)-5-amino-2-[[(2S)-2-[[(2S)-2-[[(2S,3R)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-5-amino-2-[[(2S)-1-[(2S,3R)-2-[[(2S)-2-[[(2S)-2-[[(2R)-2-[[(2S)-2-[[(2S)-2-[[2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-1-[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-amino-4-methylsulfanylbutanoyl]amino]-3-(1H-indol-3-yl)propanoyl]amino]-5-carbamimidamidopentanoyl]amino]propanoyl]pyrrolidine-2-carbonyl]amino]-3-methylbutanoyl]amino]-4-methylpentanoyl]amino]-4-methylpentanoyl]amino]acetyl]amino]-3-hydroxypropanoyl]amino]-4-methylpentanoyl]amino]-3-sulfanylpropanoyl]amino]-4-methylsulfanylbutanoyl]amino]-5-carbamimidamidopentanoyl]amino]-3-hydroxybutanoyl]pyrrolidine-2-carbonyl]amino]-5-oxopentanoyl]amino]-3-hydroxypropanoyl]amino]-3-hydroxypropanoyl]amino]-3-(1H-imidazol-5-yl)propanoyl]amino]-4-methylpentanoyl]amino]-3-hydroxybutanoyl]amino]-3-(1H-indol-3-yl)propanoyl]amino]-5-carbamimidamidopentanoyl]amino]-5-oxopentanoyl]amino]-3-hydroxybutanoyl]amino]-3-hydroxypropanoyl]amino]-3-carboxypropanoyl]amino]-3-hydroxypropanoyl]amino]-5-oxopentanoyl]amino]-5-oxopentanoyl]amino]-3-phenylpropanoyl]amino]-5-carbamimidamidopentanoyl]amino]-3-methylbutanoyl]amino]-4-methylpentanoyl]amino]-4-oxobutanoyl]amino]-5-carbamimidamidopentanoyl]amino]-3-(1H-indol-3-yl)propanoyl]amino]-4-carboxybutanoyl]amino]-5-oxopentanoyl]amino]hexanoic acid Chemical compound CSCC[C@H](N)C(=O)N[C@@H](Cc1c[nH]c2ccccc12)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(=O)N1CCC[C@H]1C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(=O)NCC(=O)N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)N[C@@H](Cc1cnc[nH]1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](Cc1c[nH]c2ccccc12)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](Cc1ccccc1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](Cc1c[nH]c2ccccc12)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCCCN)C(O)=O PGOHTUIFYSHAQG-LJSDBVFPSA-N 0.000 description 6
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 6
- 241000282472 Canis lupus familiaris Species 0.000 description 6
- 241000283973 Oryctolagus cuniculus Species 0.000 description 6
- 108090000190 Thrombin Proteins 0.000 description 6
- 230000001580 bacterial effect Effects 0.000 description 6
- 239000003153 chemical reaction reagent Substances 0.000 description 6
- 238000011156 evaluation Methods 0.000 description 6
- -1 glidants Substances 0.000 description 6
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 description 6
- 239000002609 medium Substances 0.000 description 6
- 238000007911 parenteral administration Methods 0.000 description 6
- 108020004414 DNA Proteins 0.000 description 5
- 241001052560 Thallis Species 0.000 description 5
- 125000003277 amino group Chemical group 0.000 description 5
- 239000003146 anticoagulant agent Substances 0.000 description 5
- 230000015271 coagulation Effects 0.000 description 5
- 238000005345 coagulation Methods 0.000 description 5
- 238000001962 electrophoresis Methods 0.000 description 5
- 238000006911 enzymatic reaction Methods 0.000 description 5
- 238000001976 enzyme digestion Methods 0.000 description 5
- 239000007924 injection Substances 0.000 description 5
- 238000002347 injection Methods 0.000 description 5
- 229910052759 nickel Inorganic materials 0.000 description 5
- 229940124531 pharmaceutical excipient Drugs 0.000 description 5
- 239000011780 sodium chloride Substances 0.000 description 5
- 238000006467 substitution reaction Methods 0.000 description 5
- 239000006228 supernatant Substances 0.000 description 5
- 229960004072 thrombin Drugs 0.000 description 5
- 108060005987 Kallikrein Proteins 0.000 description 4
- 102000001399 Kallikrein Human genes 0.000 description 4
- 229940127219 anticoagulant drug Drugs 0.000 description 4
- 238000005520 cutting process Methods 0.000 description 4
- 230000001419 dependent effect Effects 0.000 description 4
- 239000002552 dosage form Substances 0.000 description 4
- 108020001507 fusion proteins Proteins 0.000 description 4
- 102000037865 fusion proteins Human genes 0.000 description 4
- 230000002401 inhibitory effect Effects 0.000 description 4
- 235000015097 nutrients Nutrition 0.000 description 4
- 239000013612 plasmid Substances 0.000 description 4
- 238000003752 polymerase chain reaction Methods 0.000 description 4
- 239000000843 powder Substances 0.000 description 4
- 238000012163 sequencing technique Methods 0.000 description 4
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 4
- 206010003178 Arterial thrombosis Diseases 0.000 description 3
- 241000282414 Homo sapiens Species 0.000 description 3
- 241000699666 Mus <mouse, genus> Species 0.000 description 3
- 241000699670 Mus sp. Species 0.000 description 3
- PBCJIPOGFJYBJE-UHFFFAOYSA-N acetonitrile;hydrate Chemical compound O.CC#N PBCJIPOGFJYBJE-UHFFFAOYSA-N 0.000 description 3
- 150000008043 acidic salts Chemical class 0.000 description 3
- 239000004480 active ingredient Substances 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 125000000129 anionic group Chemical group 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 3
- 125000002843 carboxylic acid group Chemical group 0.000 description 3
- 125000002091 cationic group Chemical group 0.000 description 3
- 239000003795 chemical substances by application Substances 0.000 description 3
- 239000000839 emulsion Substances 0.000 description 3
- 239000012634 fragment Substances 0.000 description 3
- 238000001802 infusion Methods 0.000 description 3
- 238000001990 intravenous administration Methods 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 239000013595 supernatant sample Substances 0.000 description 3
- 230000009182 swimming Effects 0.000 description 3
- 239000003053 toxin Substances 0.000 description 3
- 231100000765 toxin Toxicity 0.000 description 3
- 108700012359 toxins Proteins 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 108020004705 Codon Proteins 0.000 description 2
- 108010013369 Enteropeptidase Proteins 0.000 description 2
- 241000588724 Escherichia coli Species 0.000 description 2
- 102000009123 Fibrin Human genes 0.000 description 2
- 108010073385 Fibrin Proteins 0.000 description 2
- BWGVNKXGVNDBDI-UHFFFAOYSA-N Fibrin monomer Chemical compound CNC(=O)CNC(=O)CN BWGVNKXGVNDBDI-UHFFFAOYSA-N 0.000 description 2
- 108010049003 Fibrinogen Proteins 0.000 description 2
- 102000008946 Fibrinogen Human genes 0.000 description 2
- 238000012404 In vitro experiment Methods 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 108020004511 Recombinant DNA Proteins 0.000 description 2
- 206010047249 Venous thrombosis Diseases 0.000 description 2
- 241001430591 Viridasius fasciatus Species 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- 239000000443 aerosol Substances 0.000 description 2
- 150000001447 alkali salts Chemical class 0.000 description 2
- 150000001408 amides Chemical class 0.000 description 2
- 150000001412 amines Chemical class 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 230000002785 anti-thrombosis Effects 0.000 description 2
- 230000004071 biological effect Effects 0.000 description 2
- 230000037396 body weight Effects 0.000 description 2
- 239000011575 calcium Substances 0.000 description 2
- 230000015556 catabolic process Effects 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 239000003593 chromogenic compound Substances 0.000 description 2
- 238000010367 cloning Methods 0.000 description 2
- 239000006071 cream Substances 0.000 description 2
- 238000006731 degradation reaction Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 239000006196 drop Substances 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 150000002148 esters Chemical class 0.000 description 2
- 238000000855 fermentation Methods 0.000 description 2
- 230000004151 fermentation Effects 0.000 description 2
- 229950003499 fibrin Drugs 0.000 description 2
- 229940012952 fibrinogen Drugs 0.000 description 2
- 239000000945 filler Substances 0.000 description 2
- 230000004927 fusion Effects 0.000 description 2
- 239000000499 gel Substances 0.000 description 2
- 210000004907 gland Anatomy 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 230000036541 health Effects 0.000 description 2
- 238000003780 insertion Methods 0.000 description 2
- 230000037431 insertion Effects 0.000 description 2
- 238000001361 intraarterial administration Methods 0.000 description 2
- 238000007834 ligase chain reaction Methods 0.000 description 2
- 239000012669 liquid formulation Substances 0.000 description 2
- 238000011068 loading method Methods 0.000 description 2
- 239000006210 lotion Substances 0.000 description 2
- 230000035772 mutation Effects 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 239000002674 ointment Substances 0.000 description 2
- 230000037361 pathway Effects 0.000 description 2
- 239000002244 precipitate Substances 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 230000010076 replication Effects 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 238000011895 specific detection Methods 0.000 description 2
- 239000002708 spider venom Substances 0.000 description 2
- 238000004659 sterilization and disinfection Methods 0.000 description 2
- 238000007920 subcutaneous administration Methods 0.000 description 2
- 239000000829 suppository Substances 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 238000002560 therapeutic procedure Methods 0.000 description 2
- 230000000699 topical effect Effects 0.000 description 2
- 231100000331 toxic Toxicity 0.000 description 2
- 230000002588 toxic effect Effects 0.000 description 2
- 238000013518 transcription Methods 0.000 description 2
- 230000035897 transcription Effects 0.000 description 2
- 230000002103 transcriptional effect Effects 0.000 description 2
- YDMBNDUHUNWWRP-VJBWXMMDSA-N (2s)-1-[(2r)-2-amino-3-phenylpropanoyl]-n-[(2s)-5-(diaminomethylideneamino)-1-(4-nitroanilino)-1-oxopentan-2-yl]piperidine-2-carboxamide Chemical compound C([C@@H](N)C(=O)N1[C@@H](CCCC1)C(=O)N[C@@H](CCCN=C(N)N)C(=O)NC=1C=CC(=CC=1)[N+]([O-])=O)C1=CC=CC=C1 YDMBNDUHUNWWRP-VJBWXMMDSA-N 0.000 description 1
- WPVINHLVHHPBMK-ULQDDVLXSA-N (2s)-n-[(2s)-5-(diaminomethylideneamino)-1-(4-nitroanilino)-1-oxopentan-2-yl]-1-[(2s)-5-oxopyrrolidine-2-carbonyl]pyrrolidine-2-carboxamide Chemical compound C([C@H]1C(=O)N[C@@H](CCCN=C(N)N)C(=O)NC=2C=CC(=CC=2)[N+]([O-])=O)CCN1C(=O)[C@@H]1CCC(=O)N1 WPVINHLVHHPBMK-ULQDDVLXSA-N 0.000 description 1
- KQJTXYQXRHCWKW-PJSBSAQXSA-N (4s)-4-[[(2s,3s)-2-benzamido-3-methylpentanoyl]amino]-5-[[2-[[(2s)-5-(diaminomethylideneamino)-1-(4-nitroanilino)-1-oxopentan-2-yl]amino]-2-oxoethyl]amino]-5-oxopentanoic acid;hydrochloride Chemical compound Cl.N([C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCC(O)=O)C(=O)NCC(=O)N[C@@H](CCCN=C(N)N)C(=O)NC=1C=CC(=CC=1)[N+]([O-])=O)C(=O)C1=CC=CC=C1 KQJTXYQXRHCWKW-PJSBSAQXSA-N 0.000 description 1
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- 108020000948 Antisense Oligonucleotides Proteins 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 description 1
- 108020004638 Circular DNA Proteins 0.000 description 1
- 108700010070 Codon Usage Proteins 0.000 description 1
- 102000053602 DNA Human genes 0.000 description 1
- BWGNESOTFCXPMA-UHFFFAOYSA-N Dihydrogen disulfide Chemical compound SS BWGNESOTFCXPMA-UHFFFAOYSA-N 0.000 description 1
- 208000005189 Embolism Diseases 0.000 description 1
- 206010014522 Embolism venous Diseases 0.000 description 1
- 102100029727 Enteropeptidase Human genes 0.000 description 1
- 108010014173 Factor X Proteins 0.000 description 1
- 206010062713 Haemorrhagic diathesis Diseases 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 101710138657 Neurotoxin Proteins 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 108700026244 Open Reading Frames Proteins 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 1
- 108010039286 S 2238 Proteins 0.000 description 1
- 108091081024 Start codon Proteins 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 1
- 108020005038 Terminator Codon Proteins 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 239000011149 active material Substances 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- 239000003463 adsorbent Substances 0.000 description 1
- 150000001298 alcohols Chemical class 0.000 description 1
- 208000026935 allergic disease Diseases 0.000 description 1
- 125000003368 amide group Chemical group 0.000 description 1
- 238000010640 amide synthesis reaction Methods 0.000 description 1
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 1
- 238000012870 ammonium sulfate precipitation Methods 0.000 description 1
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 1
- 229960000723 ampicillin Drugs 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 150000001450 anions Chemical class 0.000 description 1
- 230000000181 anti-adherent effect Effects 0.000 description 1
- 239000003911 antiadherent Substances 0.000 description 1
- 238000013176 antiplatelet therapy Methods 0.000 description 1
- 239000000074 antisense oligonucleotide Substances 0.000 description 1
- 238000012230 antisense oligonucleotides Methods 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 108010031969 benzoyl-Ile-Glu-Gly-Arg-p-nitroanilide Proteins 0.000 description 1
- 239000011230 binding agent Substances 0.000 description 1
- 230000017531 blood circulation Effects 0.000 description 1
- 239000006172 buffering agent Substances 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 239000001110 calcium chloride Substances 0.000 description 1
- 235000011148 calcium chloride Nutrition 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 0.000 description 1
- 239000007894 caplet Substances 0.000 description 1
- 239000002775 capsule Substances 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 150000001735 carboxylic acids Chemical class 0.000 description 1
- 150000001768 cations Chemical class 0.000 description 1
- 239000013592 cell lysate Substances 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000002738 chelating agent Substances 0.000 description 1
- 238000011098 chromatofocusing Methods 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 230000002759 chromosomal effect Effects 0.000 description 1
- 230000004087 circulation Effects 0.000 description 1
- 239000003086 colorant Substances 0.000 description 1
- 239000002299 complementary DNA Substances 0.000 description 1
- 239000012059 conventional drug carrier Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000003111 delayed effect Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 230000008034 disappearance Effects 0.000 description 1
- 239000007884 disintegrant Substances 0.000 description 1
- 208000035475 disorder Diseases 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 231100000673 dose–response relationship Toxicity 0.000 description 1
- 239000003937 drug carrier Substances 0.000 description 1
- 230000002500 effect on skin Effects 0.000 description 1
- 239000003995 emulsifying agent Substances 0.000 description 1
- 230000001804 emulsifying effect Effects 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 238000007824 enzymatic assay Methods 0.000 description 1
- 238000011067 equilibration Methods 0.000 description 1
- 210000003743 erythrocyte Anatomy 0.000 description 1
- 210000003527 eukaryotic cell Anatomy 0.000 description 1
- 238000001704 evaporation Methods 0.000 description 1
- 230000008020 evaporation Effects 0.000 description 1
- 230000007717 exclusion Effects 0.000 description 1
- 239000013613 expression plasmid Substances 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 238000013213 extrapolation Methods 0.000 description 1
- 230000006624 extrinsic pathway Effects 0.000 description 1
- 239000003889 eye drop Substances 0.000 description 1
- 229940012356 eye drops Drugs 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000000796 flavoring agent Substances 0.000 description 1
- 239000006260 foam Substances 0.000 description 1
- KANJSNBRCNMZMV-ABRZTLGGSA-N fondaparinux Chemical compound O[C@@H]1[C@@H](NS(O)(=O)=O)[C@@H](OC)O[C@H](COS(O)(=O)=O)[C@H]1O[C@H]1[C@H](OS(O)(=O)=O)[C@@H](O)[C@H](O[C@@H]2[C@@H]([C@@H](OS(O)(=O)=O)[C@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O[C@@H]4[C@@H]([C@@H](O)[C@H](O)[C@@H](COS(O)(=O)=O)O4)NS(O)(=O)=O)[C@H](O3)C(O)=O)O)[C@@H](COS(O)(=O)=O)O2)NS(O)(=O)=O)[C@H](C(O)=O)O1 KANJSNBRCNMZMV-ABRZTLGGSA-N 0.000 description 1
- 229960001318 fondaparinux Drugs 0.000 description 1
- 235000013355 food flavoring agent Nutrition 0.000 description 1
- 235000003599 food sweetener Nutrition 0.000 description 1
- 239000003349 gelling agent Substances 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- 239000003979 granulating agent Substances 0.000 description 1
- 159000000011 group IA salts Chemical class 0.000 description 1
- 208000031169 hemorrhagic disease Diseases 0.000 description 1
- 230000023597 hemostasis Effects 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 238000002513 implantation Methods 0.000 description 1
- 238000010874 in vitro model Methods 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 238000007917 intracranial administration Methods 0.000 description 1
- 238000007918 intramuscular administration Methods 0.000 description 1
- 238000010255 intramuscular injection Methods 0.000 description 1
- 239000007927 intramuscular injection Substances 0.000 description 1
- 238000007912 intraperitoneal administration Methods 0.000 description 1
- 239000007928 intraperitoneal injection Substances 0.000 description 1
- 238000007913 intrathecal administration Methods 0.000 description 1
- 238000007914 intraventricular administration Methods 0.000 description 1
- 230000006623 intrinsic pathway Effects 0.000 description 1
- 238000005342 ion exchange Methods 0.000 description 1
- 230000007794 irritation Effects 0.000 description 1
- 238000001155 isoelectric focusing Methods 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 239000002502 liposome Substances 0.000 description 1
- 239000008297 liquid dosage form Substances 0.000 description 1
- 239000007937 lozenge Substances 0.000 description 1
- 239000000314 lubricant Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 239000004005 microsphere Substances 0.000 description 1
- 239000007922 nasal spray Substances 0.000 description 1
- 239000006199 nebulizer Substances 0.000 description 1
- 239000002581 neurotoxin Substances 0.000 description 1
- 231100000618 neurotoxin Toxicity 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 229940121479 osocimab Drugs 0.000 description 1
- 239000006072 paste Substances 0.000 description 1
- 150000002989 phenols Chemical class 0.000 description 1
- 231100000614 poison Toxicity 0.000 description 1
- 239000002574 poison Substances 0.000 description 1
- 230000004481 post-translational protein modification Effects 0.000 description 1
- 230000001124 posttranscriptional effect Effects 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 210000001236 prokaryotic cell Anatomy 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 230000002685 pulmonary effect Effects 0.000 description 1
- 230000002829 reductive effect Effects 0.000 description 1
- 230000022532 regulation of transcription, DNA-dependent Effects 0.000 description 1
- 230000003362 replicative effect Effects 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 238000012807 shake-flask culturing Methods 0.000 description 1
- 238000004513 sizing Methods 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000007901 soft capsule Substances 0.000 description 1
- 238000010563 solid-state fermentation Methods 0.000 description 1
- 239000007921 spray Substances 0.000 description 1
- 238000001694 spray drying Methods 0.000 description 1
- 230000000087 stabilizing effect Effects 0.000 description 1
- 239000007929 subcutaneous injection Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 239000000375 suspending agent Substances 0.000 description 1
- 239000003765 sweetening agent Substances 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 230000009885 systemic effect Effects 0.000 description 1
- 239000003826 tablet Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000011287 therapeutic dose Methods 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 239000002562 thickening agent Substances 0.000 description 1
- 238000011200 topical administration Methods 0.000 description 1
- 239000012049 topical pharmaceutical composition Substances 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
- 230000014616 translation Effects 0.000 description 1
- 238000011269 treatment regimen Methods 0.000 description 1
- 229910021642 ultra pure water Inorganic materials 0.000 description 1
- 239000012498 ultrapure water Substances 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
- 239000002435 venom Substances 0.000 description 1
- 231100000611 venom Toxicity 0.000 description 1
- 210000001048 venom Anatomy 0.000 description 1
- 208000004043 venous thromboembolism Diseases 0.000 description 1
- 239000000080 wetting agent Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
- C07K14/43513—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from arachnidae
- C07K14/43518—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from arachnidae from spiders
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
- A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2800/00—Nucleic acids vectors
- C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/185—Escherichia
- C12R2001/19—Escherichia coli
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- General Engineering & Computer Science (AREA)
- Medicinal Chemistry (AREA)
- Biotechnology (AREA)
- Wood Science & Technology (AREA)
- Biophysics (AREA)
- Biomedical Technology (AREA)
- Insects & Arthropods (AREA)
- Veterinary Medicine (AREA)
- Physics & Mathematics (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Plant Pathology (AREA)
- Microbiology (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Hematology (AREA)
- Diabetes (AREA)
- Tropical Medicine & Parasitology (AREA)
- Toxicology (AREA)
- Gastroenterology & Hepatology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention relates to the technical field of polypeptide drug research and development. Specifically disclosed are polypeptide VKTP-Vf13, pharmaceutical compositions and uses thereof. The polypeptide is derived from zebra spiders and comprises the following polypeptides of A1 or A2: a polypeptide with an A1 amino acid sequence of SEQ ID NO. 1; the amino acid sequence A2 is polypeptide which is obtained by substituting and/or deleting and/or adding a plurality of amino acid residues in the amino acid sequence of SEQ ID NO.1, has the identity of 90% or more with the SEQ ID NO.1 and has the same function with A1. The polypeptide specifically inhibits FXIa, has little inhibition effect on FXIa and FXa, and has small bleeding risk and obvious anticoagulation effect. Has the pharmaceutical characteristics of easy preparation by a prokaryotic expression mode and good water solubility, is suitable for treating thrombus diseases, has good clinical application prospect, and can be used as candidate molecules of alternative drugs or auxiliary drugs of the existing anticoagulation drugs.
Description
Technical Field
The invention belongs to the technical field of polypeptide drug research and development, and particularly relates to a polypeptide VKTP-Vf13, a pharmaceutical composition and application thereof.
Background
The high medical costs of thromboembolic events underscores the need for newer and better treatment regimens to manage thrombotic disorders. Thrombus can be classified into arterial thrombus and venous thrombus. Arterial thrombosis tends to occur where plaque forms and where shear stress is high, forming a platelet rich "white thrombus". In contrast, in venous thrombotic disease, thrombus tends to occur at the site of damage to the vein wall, but the blood flow and shear stress are lower, resulting in a red blood cell-rich "red thrombus". Thus, anti-platelet therapy is considered to be the best option for preventing arterial thrombosis, while anticoagulant therapy is the recommended treatment for venous thrombosis. However, venous thrombosis and arterial thrombosis are not completely independent of each other, and anticoagulant therapy has a role in arterial thrombotic disease.
However, currently clinically used anticoagulants (such as sarbans, fondaparinux, and the like) mainly aim at FXa in an extrinsic pathway of a coagulation cascade, and a new generation of anticoagulants with bleeding side effects (Beavers,C.J.et al.Osocimab:A Novel Agent in Preventing Venous Thromboembolism.J Cardiovasc Pharmacol,2020,76,645-649.). are seen on an intrinsic pathway with small bleeding side effects. FXIa is taken as a key factor of an endogenous way, and the effect in thrombosis is far greater than the effect of hemostasis, so that FXIa becomes a hot target for development of a new generation of anticoagulation medicines. Currently, FXI/FXIa drugs that have entered clinical trials include small molecules, antibodies, antisense oligonucleotides and polypeptides, and early clinical efficacy is better, and the risk of bleeding is greatly reduced (Circulation 147 (2023) 897-913, 10.1161/CIRCULATIONAH.122.062353.). The above shows that FXIa has close relation with human thrombotic diseases, and inhibiting FXIa has obvious anticoagulation effect, but has no obvious bleeding tendency, and can greatly reduce the bleeding risk in the clinical anticoagulation process.
The spider venom contains a large number of toxin polypeptide molecules, has different activities and is a natural polypeptide drug molecule library. So far, the main reported active molecule is neurotoxin, and less (Luddecke,T.,et al.The biology and evolution of spider venoms.Biol Rev Camb Philos Soc,2022,97,163-178.). is studied on toxins of other active categories, so that the development of polypeptide drugs with good anticoagulation effect and small side effect has important research significance by analyzing transcriptomes of various spiders and excavating possible antithrombotic active polypeptides through specific experiments.
Disclosure of Invention
The invention aims to overcome the defects in the prior art, provides polypeptide VKTP-Vf13, a pharmaceutical composition and application thereof, and polypeptide VKTP-Vf13 can specifically inhibit FXIa, has little inhibition effect on FXIa and FXa at a certain concentration, thus having excellent anticoagulation effect, can obviously prolong the activated partial thromboplastin time (aPPT) in a concentration dependency, has no influence on Prothrombin Time (PT) at the same concentration of aPPT, and has the advantages of small bleeding side effect and obvious antithrombotic effect.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
A first object of the present invention is to provide a polypeptide VKTP-Vf13, said polypeptide VKTP-Vf13 comprising a polypeptide of A1 or A2 as follows: a polypeptide with an A1 amino acid sequence of SEQ ID NO. 1; the amino acid sequence A2 is polypeptide which is obtained by substituting and/or deleting and/or adding a plurality of amino acid residues in the amino acid sequence of SEQ ID NO.1, has the identity of 90% or more with the SEQ ID NO.1 and has the same function with A1.
It is a second object of the present invention to provide a nucleic acid molecule encoding the above polypeptide VKTP-Vf13.
Further, the nucleic acid molecules include the nucleic acid molecules shown in a1 or a2 or a3 as follows:
the a1 coding region comprises a nucleic acid molecule of SEQ ID NO. 2;
a2 nucleic acid molecule having the nucleotide sequence of SEQ ID NO. 2;
a3 has 90% or more identity to the nucleotide sequence defined in a1 or a2 and encodes a nucleic acid molecule as described above.
A third object of the present invention is to provide a recombinant vector comprising the above-mentioned nucleic acid molecule.
It is a fourth object of the present invention to provide a recombinant expression cell as described above comprising the recombinant vector as described above.
A fifth object of the present invention is to provide a method for preparing the above polypeptide VKTP-Vf13, comprising the steps of: linearizing the recombinant expression vector through enzyme cutting sites; introducing the linearized recombinant expression vector into a host cell to obtain a recombinant expression cell, culturing the recombinant expression cell, and obtaining the polypeptide VKTP-Vf13 from the culture.
A sixth object of the present invention is to provide a pharmaceutical composition comprising the polypeptide VKTP-Vf13 or a pharmaceutically acceptable salt thereof, and a pharmaceutical adjuvant.
In the pharmaceutical composition, the polypeptide VKTP-Vf13 or a pharmaceutically acceptable salt thereof may be used in an amount that is therapeutically effective.
The pharmaceutical excipients can be widely used in the field of pharmaceutical production. Adjuvants are used primarily to provide a safe, stable and functional pharmaceutical composition, and may also provide means for allowing the subject to dissolve at a desired rate after administration, or for promoting effective absorption of the active ingredient after administration of the composition. The pharmaceutical excipients may be inert fillers or provide a function such as stabilizing the overall pH of the composition or preventing degradation of the active ingredients of the composition. The pharmaceutical excipients can comprise one or more of the following excipients: binders, suspending agents, emulsifiers, diluents, fillers, granulating agents, sizing agents, disintegrants, lubricants, anti-adherents, glidants, wetting agents, gelling agents, absorption retarders, dissolution inhibitors, enhancing agents, adsorbents, buffering agents, chelating agents, preservatives, colorants, flavoring agents, and sweeteners.
The pharmaceutical compositions of the present invention may be prepared in accordance with the disclosure using any method known to those of skill in the art. For example, conventional mixing, dissolving, granulating, emulsifying, levigating, encapsulating, entrapping or lyophilizing processes, and the like.
The compositions may be formulated in unit dosage form, with each dose containing from about 5 to 1000mg, more typically from about 100 to 500mg, of the active ingredient. The term "unit dosage form" refers to physically discrete unitary dosage units suitable as unitary dosages for human subjects and other mammals, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical excipient.
The effective dosage of the active agent can range widely, and is typically administered in a pharmaceutically effective amount. It will be appreciated that the amount of the compound actually administered will generally be determined by the physician, in light of the relevant circumstances, and will include the condition to be treated, the chosen route of administration, the actual compound administered; age, weight, and response of the individual patient; severity of patient symptoms, and the like.
The pharmaceutical compositions of the present invention may be administered in any form, including injection (intravenous), mucosal, oral (solid and liquid formulations), inhalation, ocular, rectal, topical or parenteral (infusion, injection, implantation, subcutaneous, intravenous, intra-arterial, intramuscular). The pharmaceutical compositions of the invention may also be in controlled or delayed release dosage forms (e.g., liposomes or microspheres). Examples of solid oral formulations include, but are not limited to, powders, capsules, caplets, soft capsules, and tablets. Examples of liquid formulations for oral or mucosal administration include, but are not limited to, suspensions, emulsions, elixirs and solutions. Examples of topical formulations include, but are not limited to, emulsions, gels, ointments, creams, patches, pastes, foams, lotions, drops or serum formulations. Examples of formulations for parenteral administration include, but are not limited to, solutions for injection, dry formulations which may be dissolved or suspended in a pharmaceutically acceptable carrier, suspensions for injection, and emulsions for injection. Examples of other suitable formulations of the pharmaceutical composition include, but are not limited to, eye drops and other ophthalmic formulations; aerosol: such as nasal sprays or inhalants; a liquid dosage form suitable for parenteral administration; suppositories and lozenges.
The seventh object of the present invention is to provide an application of the polypeptide VKTP-Vf13 or a pharmaceutically acceptable salt thereof in preparing FXIa inhibitor.
An eighth object of the present invention is to provide the use of the above polypeptide VKTP-Vf13 or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for treating FXIa-related diseases.
A ninth object of the present invention is to provide an application of the polypeptide VKTP-Vf13 or a pharmaceutically acceptable salt thereof in preparing a medicament for treating a disease associated with blood coagulation, the disease associated with blood coagulation is a thrombus, and the thrombus is a white thrombus, a red thrombus, a mixed thrombus or a transparent thrombus.
When used as a medicament, the polypeptides of the invention may be administered in the form of a pharmaceutical composition. These compositions may be prepared in a manner well known in the pharmaceutical arts and may be administered by a variety of routes, depending upon whether local or systemic treatment and the area being treated is desired. Topical (e.g., transdermal, dermal, ocular, and mucosal including intranasal, vaginal, and rectal delivery), pulmonary (e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal), oral, or parenteral administration. Parenteral administration includes intravenous, intra-arterial, subcutaneous, intraperitoneal, or intramuscular injection or infusion; or intracranial, e.g., intrathecal or intraventricular, administration. Parenteral administration may be in the form of a single bolus dose or may be administered by, for example, a continuous infusion pump. Pharmaceutical compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, water, powder or oily matrices, thickeners and the like may be necessary or desirable.
Therapeutic doses of the polypeptides of the invention may be determined, for example, according to the following: the specific use of the treatment, the manner in which the compound is administered, the health and condition of the patient, and the discretion of the prescribing physician. The proportion or concentration of the polypeptide of the invention in the pharmaceutical composition may be variable, depending on a number of factors, including the dosage, chemical nature and route of administration. The compounds of the invention may be provided, for example, by a physiologically buffered aqueous solution containing about 0.1 to 10% w/v of the compound for parenteral administration. Some typical dosages range from about 1 μg/kg to about 1g/kg body weight/day. In certain embodiments, the dosage ranges from about 0.01mg/kg to about 100mg/kg body weight/day. Dosages will likely depend on such variables as the type and extent of progression of the disease or disorder, the general health of the particular patient, the relative biological efficacy of the compound selected, the excipient formulation and its route of administration. The effective dose can be obtained by extrapolation of a dose response curve derived from in vitro or animal model test systems.
The term "identity" as used in the present invention refers to identity of amino acid sequences or nucleotide sequences. The identity of amino acid sequences can be determined using homology search sites on the internet, such as BLAST web pages of the NCBI homepage website. For example, in advanced BLAST2.1, by using blastp as a program, expect values are set to 10, all filters are set to OFF, BLOSUM62 is used as matrix, gap existence cost, per residue gap cost and lambda ratio are set to 11,1 and 0.85 (default values), respectively, and identity of a pair of amino acid sequences is searched for and calculated, and then the value (%) of identity can be obtained.
The term "protein having the same function" as used in the present invention has the same or similar meaning as conventionally understood by those skilled in the art, and means that the amino acid sequence of a fragment is a part of the amino acid sequence of an intact protein or polypeptide, and has the same or similar function or activity as the intact protein or polypeptide. It will be appreciated by those of ordinary skill in the art that altering a minority of amino acid residues in certain regions of a polypeptide, e.g., non-important regions, does not substantially alter biological activity, e.g., the sequence resulting from appropriate substitution of certain amino acids does not affect its activity (see Watson et al Molecular Biologyof The Gene, fourth edition, 1987,The Benjamin/Cummings pub. Co. P224). Thus, one of ordinary skill in the art would be able to perform such substitutions and ensure that the resulting molecule still has the desired biological activity.
The term "encoding nucleic acid molecule" as used in the present invention means a polynucleotide directly specifying the amino acid sequence of a polypeptide. The boundaries of the coding sequence are generally determined by an open reading frame beginning with a start codon (e.g., ATG, GTG or TTG) and ending with a stop codon (e.g., TAA, TAG or TGA). The coding sequence may be genomic DNA, synthetic DNA, or a combination thereof.
The term "expression" as used in the present invention includes any step involving the production of a polypeptide, including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.
The term "recombinant vector" as used in the present invention means a linear or circular DNA molecule comprising a polynucleotide encoding a polypeptide operably linked to control sequences for its expression.
The term "host cell" as used in the present invention means any cell type that is readily transformed, transfected, transduced, or the like with a nucleic acid construct or expression vector comprising a polynucleotide of the present invention. The term "host cell" encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication.
The term "pharmaceutically acceptable" as used herein is intended to refer to those compounds, materials, compositions and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
The term "pharmaceutically acceptable salt" refers to a salt-forming reaction conventional in the art, such as: salts are formed from chemical reactions between bases and acids, such as: NH 3+H2SO4→(NH4)2SO4.
The salt may be a basic salt, an acidic salt, or a neutral salt. The basic salt generates hydroxide ions in the water and the acidic salt generates hydronium ions.
Polypeptide VKTP-Vf13 may form a salt of polypeptide VKTP-Vf13 of the invention with a cation or anion between anionic groups or cationic groups, respectively. These groups may be located in the peptide portion of the polypeptide VKTP-Vf13 of the invention.
The anionic group of polypeptide VKTP-Vf13 of the present invention may include the free carboxyl group of the peptide moiety. The peptide moiety typically includes a free carboxylic acid group at the C-terminus.
The cationic group of the peptide moiety is not limited in the present invention and includes the free amino group at the N-terminus (if present) as well as any free amino groups of internal basic amino acid residues (e.g., arg and Lys).
In a specific embodiment, the analog of polypeptide VKTP-Vf13 of the invention is an alkaline salt. These salts may be formed, for example, between the anionic groups of the peptide moiety and sodium or potassium cations.
In another embodiment, the analog of polypeptide VKTP-Vf13 of the invention is an acidic salt. These salts may be formed, for example, between the cationic groups of the peptide moiety and chloride or acetate anions.
The free carboxylic acid groups may also be reacted with alcohols or phenols to form esters of the derivatives of the invention, which may involve free carboxylic groups at the C-terminus of the peptide and/or any free carboxylic groups in the side chains.
Amides of the derivatives of the invention may also be formed by reacting free carboxylic acid groups with amines or substituted amines, or by reacting free or substituted amino groups with carboxylic acids. Amide formation may involve free carboxyl groups at the C-terminus of the peptide, any free carboxyl groups in the side chains, free amino groups at the N-terminus of the peptide and/or any free or substituted amino groups of the peptide and/or the peptide in the side chains.
In a specific embodiment, the polypeptide VKTP-Vf13 is in the form of a pharmaceutically acceptable salt. In another specific embodiment, the polypeptide VKTP-Vf13 is in the form of a pharmaceutically acceptable amide, preferably with an amide group at the C-terminus of the peptide. In yet another specific embodiment, the polypeptide VKTP-Vf13 is in the form of a pharmaceutically acceptable ester.
Compared with the prior art, the technical scheme provided by the invention has the beneficial effects that:
(1) According to the invention, the zebra spider (Viridasius fasciatus) is taken as a research object, and the polypeptide VKTP-Vf13 derived from the zebra spider and the encoding gene thereof are discovered for the first time through sequencing a transcriptome of a poison gland sample, analyzing data and selecting the polypeptide, so that the polypeptide has remarkable inhibition effect on FXIa, and has the characteristics of small bleeding risk, good anticoagulation effect and the like.
(2) The polypeptide VKTP-Vf13 (SACDLPAETGVCRGFFPSYYFDSTTGQCQKFVYGGCGGNDNNFETVEQCQESCAAS) with the amino acid sequence of SEQ ID NO.1 is efficiently prepared by constructing recombinant plasmid and recombinant cell expression, the molecular weight of the polypeptide is 5985.46Da, and the polypeptide consists of 56 amino acid residues and 3 pairs of disulfide bonds.
(3) According to the invention, through carrying out research on inhibiting FXIa, FXIa and FXa enzyme activities on polypeptide VKTP-Vf13, research results show that the polypeptide has obvious concentration-dependent inhibition on FXIa enzyme, the half inhibition concentration is about 99.81+/-8.10 nM, the polypeptide hardly has inhibition effect on FXIa and FXa enzyme, and meanwhile, the polypeptide hardly has inhibition effect on Thrombin and KALLIKREIN enzyme; the effect of polypeptide VKTP-Vf13 on the activated partial thromboplastin time of mouse, dog, and rabbit plasma was examined by in vitro experiments. The results show that polypeptide VKTP-Vf13 can extend the aPPT in mice, dogs, and rabbits in a concentration-dependent manner; the effect of polypeptide VKTP-Vf13 on prothrombin time in mouse, dog and rabbit plasma was examined by in vitro experiments. The results show that polypeptide VKTP-Vf13 had no significant effect on PT in mice, dogs and rabbits.
(4) The invention obtains a novel FXIa inhibitor, can be used for treating diseases related to blood coagulation, and preparing medicaments for treating the diseases or the diseases, provides a brand new lead polypeptide molecule for developing novel anticoagulation medicaments, provides a method reference for other toxic animal resources without developing active polypeptides, and can be used as candidate molecules of alternative medicaments or auxiliary medicaments of the existing anticoagulation medicaments.
Drawings
FIG. 1 is a schematic diagram of a 3D structure and disulfide bond pairing scheme of a polypeptide VKTP-Vf13 provided by the present invention;
FIG. 2 is a graph showing the identification result of recombinant expression plasmid VKTP-Vf13-PET-32a (+) constructed in the present invention;
FIG. 3 is a diagram showing the SDS-PAGE identification of the polypeptide VKTP-Vf13 of the present invention; swimming band 1: bacterial liquid supernatant; swimming band 2: fusion protein VKTP-Vf13; swimming band 3: degradation of the fusion protein VKTP-Vf13;
FIG. 4 is a graph showing the trend of polypeptide VKTP-Vf13 of the present invention to inhibit FXIa enzyme activity;
FIG. 5 is a graph of half maximal inhibitory concentration (IC 50) of polypeptide VKTP-Vf13 of the invention inhibiting FXIa activity;
FIG. 6 is a graph showing the effect of polypeptide VKTP-Vf13 of the present invention on various protease activities;
FIG. 7 is a graph showing the evaluation of the effect of polypeptide VKTP-Vf13 of the present invention on activated partial thromboplastin time (aPPT);
FIG. 8 is a graph showing the evaluation of the effect of the polypeptide VKTP-Vf13 of the present invention on Prothrombin Time (PT).
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the following detailed description of the specific embodiments of the present invention will be given with reference to the accompanying drawings. The examples are not to be construed as limiting the specific techniques or conditions described in the literature in this field or as per the specifications of the product. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
One embodiment provided by the invention relates to polypeptide VKTP-Vf13, wherein the polypeptide has at least 90%, e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID No. 1.
A polynucleotide encoding a polypeptide VKTP-Vf13 as described above. In one embodiment, the polynucleotide encoding a polypeptide of the invention has been isolated. Techniques for isolating or cloning polynucleotides are known in the art and include isolation from genomic DNA or cDNA or a combination thereof. Cloning of polynucleotides from genomic DNA can be accomplished, for example, by using the well-known Polymerase Chain Reaction (PCR) or antibody screening of expression libraries to detect cloned DNA fragments having shared structural features. See, e.g., innis et al, 1990, PCR: AGuidetomethodsandApplication [ PCR: methods and application guide ], academic Press (academic Press), new York. Other nucleic acid amplification procedures such as Ligase Chain Reaction (LCR), ligation Activated Transcription (LAT) and polynucleotide-based amplification (NASBA) may be used.
In some embodiments, modification of a polynucleotide encoding a polypeptide of the invention may be necessary for synthesis of a polypeptide substantially similar to the polypeptide. The term "substantially similar" to the polypeptide refers to a non-naturally occurring form of the polypeptide. These polypeptides may differ from polypeptides isolated from their natural sources by some engineering means, such as variants that differ in specific activity, thermostability, pH optimum, etc. Variants can be constructed as follows: based on the polynucleotide proposed as a mature polypeptide coding sequence (e.g. a subsequence thereof), and/or by introducing nucleotide substitutions that do not result in a change in the amino acid sequence of the polypeptide, but which correspond to codon usage of a host organism intended for the production of the enzyme, or by introducing nucleotide substitutions that may result in a different amino acid sequence.
The invention also relates to recombinant vectors for constructing the above-described nucleic acid molecules, which comprise a polynucleotide of the invention operably linked to one or more control sequences that direct the expression of the coding sequence in a suitable host cell under conditions compatible with the control sequences. The polynucleotides can be manipulated in a number of ways to provide for expression of polypeptides. Depending on the expression vector, it may be desirable or necessary to manipulate the polynucleotide prior to insertion into the vector. Techniques for modifying polynucleotides using recombinant DNA methods are known in the art. The control sequence may be a promoter, i.e., a polynucleotide recognized by a host cell for expression of a polynucleotide encoding a polypeptide of the invention. The promoter contains transcriptional control sequences that mediate the expression of the polypeptide. The promoter may be any polynucleotide that exhibits transcriptional activity in the host cell including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell.
In some embodiments, it also relates to recombinant expression vectors comprising a polynucleotide of the invention, a promoter, and transcriptional and translational stop signals. Multiple nucleotides and control sequences may be linked together to produce a recombinant expression vector, which may include one or more convenient restriction sites to allow for insertion or substitution of the polynucleotide encoding the polypeptide at these sites. Alternatively, the polynucleotide may be expressed by inserting the polynucleotide or a nucleic acid construct comprising the polynucleotide into an appropriate vector for expression. In generating the expression vector, the coding sequence is located in the vector such that the coding sequence is operably linked to the appropriate control sequences for expression. The recombinant expression vector may be any vector (e.g., a plasmid or virus) that can be conveniently subjected to recombinant DNA procedures and that can cause expression of the polynucleotide. The choice of vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced. The vector may be a linear or closed circular plasmid.
In some embodiments, it also relates to recombinant host cells comprising a polynucleotide of the invention operably linked to one or more control sequences that direct the production of a polypeptide of the invention. The construct or vector comprising the polynucleotide is introduced into a host cell such that the construct or vector is maintained as a chromosomal integrant or as an autonomously replicating extra-chromosomal vector, as described earlier. The term "host cell" encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication. The choice of host cell will depend to a large extent on the gene encoding the polypeptide and its source. The host cell may be any cell useful in the recombinant production of the polypeptides of the invention, such as a prokaryotic cell or a eukaryotic cell.
The invention also relates to a method for preparing the polypeptide, wherein the recombinant expression (host) cell of the invention is cultured under the condition of benefiting the production of the polypeptide, and the polypeptide is obtained from the culture. These host cells are cultured in a nutrient medium suitable for producing the polypeptide using methods known in the art. For example, the cells may be cultured by shake flask culture, or small-scale or large-scale fermentation (including continuous, batch, fed-batch, or solid state fermentations) in laboratory or industrial fermentors performed in a suitable medium and under conditions allowing the polypeptide to be expressed and/or isolated. Culturing occurs in a suitable nutrient medium comprising carbon and nitrogen sources and inorganic salts using procedures known in the art. Suitable media are available from commercial suppliers or may be prepared according to published compositions (e.g., in catalogues of the China center for type culture Collection). If the polypeptide is secreted into the nutrient medium, the polypeptide can be recovered directly from the medium. If the polypeptide is not secreted, it can be recovered from the cell lysate.
In particular embodiments, the polypeptides may be detected using methods known in the art specific for the polypeptides. These detection methods include, but are not limited to, the use of specific antibodies, the formation of enzyme products, or the disappearance of enzyme substrates. For example, an enzymatic assay may be used to determine the activity of a polypeptide.
In particular embodiments, the polypeptides may be recovered using methods known in the art. For example, the polypeptide may be recovered from the nutrient medium by conventional methods including, but not limited to, collection, centrifugation, filtration, extraction, spray drying, evaporation, or precipitation. In another aspect, fermentation broth comprising the polypeptide may also be recovered.
In particular implementations, the polypeptides can be purified to obtain substantially pure polypeptides by a variety of methods known in the art, including, but not limited to, chromatography (e.g., ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion), electrophoretic methods (e.g., preparative isoelectric focusing), differential solubility (e.g., ammonium sulfate precipitation), SDSPAGE, or extractive purification.
The invention provides a pharmaceutical composition comprising polypeptide VKTP-Vf13 or pharmaceutically acceptable salt thereof and pharmaceutical excipients.
The polypeptide VKTP-Vf13 or a pharmaceutically acceptable salt thereof according to the invention can also be used in the preparation of FXIa inhibitors. The polypeptide VKTP-Vf13 or the pharmaceutically acceptable salt thereof can be used for preparing medicines for treating diseases related to blood coagulation, wherein the diseases related to blood coagulation are thrombus, and the thrombus can be white thrombus, red thrombus, mixed thrombus or transparent thrombus.
Example 1
1. Preparation of polypeptide VKTP-Vf 13:
(1) Discovery of polypeptide VKTP-Vf13 fragment sequence
Dissected zebra spiders (Viridasius fasciatus) toxic glands were rapidly stored in RNA preservation (BL 621A, biosharp) and sent to beijing norelsen technologies, inc. After the original off-machine data were obtained, the specific analysis method of toxin polypeptide was performed by the technology disclosed in chinese patent (CN 116486915A). Finally, the coding gene and the amino acid sequence of the polypeptide VKTP-Vf13 (Venom Kunitz Type-LIKE PEPTIDE-Vf 13) are obtained. The polypeptide VKTP-Vf13 mature peptide contains 56 amino acids. The 3D structure of polypeptide VKTP-Vf13 was obtained by on-line software SWISS-MODEL by homology modeling. The specific manner in which polypeptide VKTP-Vf13 disulfide pairs are determined by homology modeling of the 3D structure.
As shown in FIG. 1, the specific pairing of the disulfide bonds of polypeptide VKTP-Vf13 is C5-C53, C12-C36, C28-C49.
(2) Recombinant expression of polypeptide VKTP-Vf13
The VKTP-Vf13 amino sequence was optimized by on-line codon optimization software (https:// www.novopro.cn/tools/codon-optimization. Html) (see sequence SEQ ID NO: 4). An enterokinase enzyme cutting site (GACGACGACGACAAG) and a termination codon (TAA) are added at the front end and the tail end of the optimized nucleotide sequence for encoding the polypeptide VKTP-Vf13, and an EcoRI/HindIII enzyme cutting site is added at the front end and the tail end of the nucleotide sequence respectively. The above sequences were constructed by artificial synthesis (done by Nanjing gold St Biotechnology Co., ltd.) and by EcoRI/HindIII cleavage sites into the pET32a vector (see FIG. 2). BL21 (DE 3) escherichia coli is transformed by the constructed VKTP-Vf13-pET32a plasmid, monoclonal is selected and inoculated into LB culture medium containing ampicillin resistance (ST 007, biyun) for 6-8 hours at the temperature of 37 ℃ and 220rpm, split charging 300 mu L and sending to the engine organism company for sequencing, and subsequent prokaryotic expression is carried out by utilizing bacterial liquid with correct sequencing. Sequencing correctly, and recovering bacterial liquid according to the ratio of 1:100 is inoculated in 1L LB culture medium containing resistance, cultured in a shaking table at 37 ℃ and 220rpm, when the OD 600nm value reaches 0.6-0.8, IPTG with the final concentration of 1mM is added to induce the polypeptide to express, and the induced expression is carried out at 28 ℃ and 100rpm for 12-16 hours. After induction, the bacterial liquid is collected centrifugally, washed twice by ultrapure water and the supernatant is removed centrifugally. And (3) re-suspending the thalli by using PBS until no obvious thalli precipitate exists, pouring the thalli liquid into a homogenizer for continuous crushing for 3 times, ensuring that no obvious precipitate exists in the thalli liquid, and centrifugally collecting the bacterial liquid supernatant.
(3) Polypeptide VKTP-Vf13 separation and purification
Loading the pretreated bacterial liquid supernatant sample into a nickel column after balancing, eluting the hybrid protein by using 10mM and 30mM imidazole solution, eluting the target protein by using 300mM imidazole solution, and collecting. In 25mM Tris-HCl (pH8.0) system, enzyme digestion is carried out at 25 ℃ for 12-14 hours by adding 0.2U enterokinase (P4237-1000U, biyun) per 0.1mg Flag fusion protein. And crushing the thalli, centrifuging to obtain a supernatant sample, purifying the supernatant sample by a nickel column, and carrying out SDS-PAGE electrophoresis analysis on the sample after enzyme digestion. After analysis, the polypeptide sample after enzyme digestion is separated and purified by C18 reverse self-loading column. The specific conditions are as follows: the column was equilibrated with 5% acetonitrile water of 0.1% TFA, the sample after digestion was applied to the column after equilibration, 2 column volumes of 0.1% TFA of 5% acetonitrile water were desalted and washed, and one column volume of 0.1% TFA of 40% acetonitrile water eluted the protein of interest.
As shown in FIG. 3, electrophoresis of the post-sterilization supernatant, the post-nickel column purified and the post-enzyme cut samples are shown, wherein lane 1 is the post-sterilization supernatant, lane 2 is the post-nickel column purified and post-enzyme cut sample, lane 3 is the post-nickel column purified and post-enzyme cut sample, arrows in lanes 1 and 2 indicate target proteins with fusion heads, and the upper and lower arrows in lane 3 indicate fusion heads and polypeptides VKTP-Vf13 after enzyme cutting, respectively, wherein M is shown as a protein electrophoresis standard. According to the invention, a prokaryotic expression mode is adopted, the target polypeptide is efficiently expressed in escherichia coli expression bacterium BL21 through pET32a, the expression condition of the recombinant polypeptide is analyzed by SDS-PAGE electrophoresis after TEV enzyme digestion, and two bands of obvious fusion protein and the target polypeptide can be observed in a lane 3, so that the enzyme digestion is proved to be successful.
Example 2
Evaluation of FXIa Activity by polypeptide VKTP-Vf 13.
In a 96-well plate (2481, corning), 1. Mu.L of the sample was mixed with 1. Mu.L of hFXIa (HFXIa 1111a,Enzyme research) (final concentration 4.65 mU/mL) in 58. Mu.L of buffer (10 mM Tris-HCl, 150mM NaCl, 10mM MgCl 2、1mMCaCl2, 0.1% BSA, pH 7.4). After standing at room temperature for 5 minutes, 36. Mu.L of a mixture of buffer and 4. Mu.L of chromogenic substrate S-2366 (82109039, chromagenix) (concentration 0.4 mM) was added to give a final volume of 100. Mu.L. The kinetics of the enzyme reaction was monitored for a total of 30 minutes at 40 seconds intervals using an enzyme-labeled instrument (Epoch, bioTek) to detect OD 405nm absorbance.
The results of the experiment showed that polypeptide VKTP-Vf13 inhibited FXIa enzyme activity in a concentration-dependent manner in the enzyme kinetic experiment (as shown in fig. 4). According to the concentration relation of the polypeptide VKTP-Vf13 in FXIa enzyme dynamics, nonlinear fitting is carried out to calculate that half inhibition concentrations of the polypeptide VKTP-Vf13 to FXIa are 99.81+/-8.10 nM respectively (shown in figure 5).
Example 3
Evaluation of FXa Activity by polypeptide VKTP-Vf 13.
In a 96-well plate, 1. Mu.L of the sample was mixed with 1. Mu.L of FXa (HFXa 1011,Enzyme research) (final concentration 11.36 mU/mL)) in 58. Mu.L of buffer (100 mM Tris-HCl, 200mM NaCl, 0.1% BSA, pH 7.4). After 5 minutes of standing at room temperature, 36. Mu.L of a mixture of buffer and 4. Mu.L of chromogenic substrate S-2222 (82031639, chromagenix) (final concentration 0.4 mM) was added to a final volume of 100. Mu.L. The kinetics of the enzymatic reaction was monitored for a total of 30 minutes at 40 seconds intervals using a microplate reader to detect the OD 405nm absorbance.
The results of the experiment show that 2. Mu.M of polypeptide VKTP-Vf13 had no effect on FXa enzyme activity in the enzyme kinetic experiment (as shown in FIG. 6).
Example 4
Evaluation of FXIa Activity by polypeptide VKTP-Vf 13.
In a 96-well plate (2481, corning), 1. Mu.L of the sample was mixed with 1. Mu.L of FXIIIa (HFXIIa 1212a,Enzyme research) (final concentration 36.5 mU/mL) in 58. Mu.L of buffer (50 mM Tris-HCl, 100mM NaCl, 5mM CaCl2, 0.1% BSA, pH 8.0). After 5 minutes of standing at room temperature, 36. Mu.L of a mixture of buffer and 4. Mu.L of chromogenic substrate S-2302 (82034039, chromagenix) (concentration 0.2 mM) was added to give a final volume of 100. Mu.L. The kinetics of the enzymatic reaction was monitored for a total of 30 minutes at 40 seconds intervals using an enzyme-labeled instrument (Epoch, bioTek) to detect OD 405nm absorbance.
The results of the experiment show that 2. Mu.M of polypeptide VKTP-Vf13 had no effect on FXIIIa enzyme activity in the enzyme kinetic experiment (as shown in FIG. 6).
Example 5
Polypeptide VKTP-Vf13 was evaluated for Thrombin activity.
In a 96-well plate (2481, corning), 1. Mu.L of the sample was mixed with 1. Mu.L of Thrombin (HT 1002a,Enzyme research) (final concentration 10.01 mU/mL) in 58. Mu.L of buffer (100 mM Tris-HCl, 200mM NaCl, 0.1% BSA, pH 7.4). After 5 minutes of standing at room temperature, 36. Mu.L of a mixture of buffer and 4. Mu.L of chromogenic substrate S-2238 (82032939, chromagenix) (concentration 0.4 mM) was added to give a final volume of 100. Mu.L. The kinetics of the enzyme reaction was monitored for a total of 30 minutes at 40 seconds intervals using an enzyme-labeled instrument (Epoch, bioTek) to detect OD 405nm absorbance.
The experimental results show that VKTP-Vf13 in the 2 μ experiment had no effect on Thrombin enzyme activity in the enzyme kinetic experiment (as shown in fig. 6).
Example 6
Evaluation of Activity of polypeptide VKTP-Vf13 on KALLIKREIN.
In a 96-well plate, 1. Mu.L of the sample was mixed with 1. Mu.L of KALLIKREIN (HPKa 1303,Enzyme research) (final concentration 3.18 mU/mL)) in 58. Mu.L of buffer (50 mM Tris-HCl, 250mM NaCl, 0.1% BSA, pH 7.5). After 5 minutes of standing at room temperature, 36. Mu.L of a mixture of buffer and 4. Mu.L of chromogenic substrate S-2302 (82034039, chromagenix) (final concentration 0.4 mM) was added to give a final volume of 100. Mu.L. The kinetics of the enzymatic reaction was monitored for a total of 30 minutes at 40 seconds intervals using a microplate reader to detect the OD 405nm absorbance.
The experimental results show that VKTP-Vf13 at 2 μm had no effect on KALLIKREIN enzyme activity in the enzyme kinetic experiments (as shown in fig. 6).
Example 7
Polypeptide VKTP-Vf13 was evaluated for its effect on the time to activate partial thromboplastin (apt).
The activated partial thromboplastin time (app pt) experiment primarily evaluates the effect of a drug on the intrinsic coagulation pathway. In an aPPT experiment, the plasma to be measured is added with a partial thromboplastin solution, fibrinogen is converted into insoluble fibrin under the participation of Ca 2+, and the time required for coagulation is measured, namely the time for activating partial prothrombin of the plasma to be measured. The specific detection operation is carried out according to the instruction of a kit (01020138, solar organism), and the process is as follows: the APTT reagent is preheated at 37 ℃, and the APTT reagent is gently mixed in an inverted mode. After mixing 50. Mu.L of APTT reagent, 50. Mu.L of normal plasma and 5. Mu.L of sample, incubating for 5 minutes in a water bath at 37 ℃, adding 50. Mu.L of preheated CaCl 2 solution, immediately mixing, and detecting the absorbance of OD 650nm by an enzyme-labeled instrument.
As shown in FIG. 7, the results show that polypeptide VKTP-Vf13 can extend the aPPT in mice, dogs, and rabbits in a concentration-dependent manner.
Example 5
Polypeptide VKTP-Vf13 was evaluated for its effect on Prothrombin Time (PT).
Prothrombin Time (PT) experiments mainly evaluate the effect of drugs on extrinsic coagulation pathways. In PT experiments, the plasma to be measured is added with excessive amount of calcium-containing tissue thromboplastin, the recalcified plasma activates factor X to become Xa in the presence of tissue factor, the latter converts prothrombin to thrombin, thrombin converts fibrinogen to insoluble fibrin, and the time required for coagulation is measured, namely the prothrombin time of the plasma to be measured. The specific detection procedure was performed as described in the kit (01020139, sun organism), and was approximately as follows: prothrombin reagent was preheated for 15 min at 37 ℃.50 μl of normal plasma was incubated with 5 μl of sample in a 37 ℃ incubator for 5 minutes, 100 μl of pre-heated prothrombin reagent was added, and the absorbance of OD 650nm was immediately started with a microplate reader.
As shown in FIG. 8, polypeptide VKTP-Vf13 had no effect on PT in mouse, dog, and rabbit plasma.
The embodiments described above and features of the embodiments herein may be combined with each other without conflict.
The foregoing description of the preferred embodiments of the invention is not intended to limit the invention to the precise form disclosed, and any such modifications, equivalents, and alternatives falling within the spirit and scope of the invention are intended to be included within the scope of the invention.
Claims (10)
1. A polypeptide VKTP-Vf13, characterized in that: the polypeptide VKTP-Vf13 includes the polypeptide of A1 or A2: a polypeptide with an A1 amino acid sequence of SEQ ID NO. 1; the amino acid sequence A2 is polypeptide which is obtained by substituting and/or deleting and/or adding a plurality of amino acid residues in the amino acid sequence of SEQ ID NO.1, has the identity of 90% or more with the SEQ ID NO.1 and has the same function with A1.
2. A nucleic acid molecule encoding the polypeptide VKTP-Vf13 of claim 1.
3. The nucleic acid molecule of claim 2, wherein the nucleic acid molecule comprises a nucleic acid molecule as set forth in a1 or a2 or a 3:
the a1 coding region comprises a nucleic acid molecule of SEQ ID NO. 2;
a2 nucleic acid molecule having the nucleotide sequence of SEQ ID NO. 2;
a3 has 90% or more identity to the nucleotide sequence defined in a1 or a2 and encodes the nucleic acid molecule of claim 2.
4. A recombinant vector comprising the nucleic acid molecule of any one of claims 2-3.
5. A recombinant expression cell comprising the recombinant vector of claim 4.
6. A method of producing a polypeptide VKTP-Vf13 as claimed in claim 1, comprising the steps of: linearizing the recombinant expression vector of claim 4 by means of an enzyme cleavage site; introducing the linearized recombinant expression vector into a host cell to obtain a recombinant expression cell, culturing the recombinant expression cell, and obtaining the polypeptide VKTP-Vf13 from the culture.
7. A pharmaceutical composition comprising a polypeptide VKTP-Vf13 or a pharmaceutically acceptable salt thereof as claimed in claim 1, and a pharmaceutically acceptable adjuvant.
8. Use of a polypeptide VKTP-Vf13 or a pharmaceutically acceptable salt thereof according to claim 1 in the preparation of an FXIa inhibitor.
9. Use of a polypeptide VKTP-Vf13 or a pharmaceutically acceptable salt thereof as claimed in claim 1 in the manufacture of a medicament for the treatment of a disease associated with FXIa.
10. Use of a polypeptide VKTP-Vf13 or a pharmaceutically acceptable salt thereof as claimed in claim 1 in the manufacture of a medicament for the treatment of a disease associated with blood coagulation, said disease being a thrombus, said thrombus being a white thrombus, a red thrombus, a mixed thrombus or a clear thrombus.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311742628.4A CN117903276A (en) | 2023-12-15 | 2023-12-15 | Polypeptide VKTP-Vf13, pharmaceutical composition and use thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311742628.4A CN117903276A (en) | 2023-12-15 | 2023-12-15 | Polypeptide VKTP-Vf13, pharmaceutical composition and use thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN117903276A true CN117903276A (en) | 2024-04-19 |
Family
ID=90682984
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202311742628.4A Pending CN117903276A (en) | 2023-12-15 | 2023-12-15 | Polypeptide VKTP-Vf13, pharmaceutical composition and use thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117903276A (en) |
-
2023
- 2023-12-15 CN CN202311742628.4A patent/CN117903276A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US20210015900A1 (en) | Gla domains as therapeutic agents | |
| EP0370036B1 (en) | Modified factor vii/viia | |
| US9611307B2 (en) | Method for increasing granulocyte number in a patient by administering superactive IL-33 fragments | |
| US20240132573A1 (en) | Modified serpins for the treatment of bradykinin-mediated disease | |
| EP3595703A1 (en) | Recombinant mature complement factor i | |
| AU4808190A (en) | Recombinant proteins with adipsin and complement d activities | |
| US20220356455A1 (en) | Solubilized apyrases, methods and use | |
| US9127075B2 (en) | Analgesic active peptide VGG, preparation and use thereof | |
| JP2001238683A (en) | Method for microbially producing protein with furin and cell for producing the same | |
| BG63276B1 (en) | Method for the preparation of a modified inhibitor palidipine of collagen-induced thrombocyte accumulation | |
| CN117903276A (en) | Polypeptide VKTP-Vf13, pharmaceutical composition and use thereof | |
| KR20020073496A (en) | Nucleic acids encoding (poly)peptides having chips activity | |
| US7335758B2 (en) | Catalytic domain of ADAM33 and methods of use thereof | |
| JPH03503843A (en) | Method for purifying phospholipase A2 and method for producing phospholipase A2-like polypeptide | |
| CN117126268A (en) | Polypeptide Acftoxin-Tv1, pharmaceutical composition and application thereof | |
| NO317661B1 (en) | Thrombin-activatable plasminogen analogs, use of the compounds in the preparation of a pharmaceutical agent, compositions comprising the compounds, and nucleic acid | |
| WO2008046256A1 (en) | Haemocoagulase | |
| US10287338B2 (en) | Factor VIII protein compositions and methods of treating of hemophilia A | |
| NZ751494B2 (en) | Gla domains as therapeutic agents | |
| NZ710958B2 (en) | Gla domains as therapeutic agents | |
| NZ751491B2 (en) | Gla domains as targeting agents |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |