CN114437182B - 聚乙二醇化f多肽及其制备方法与用途 - Google Patents
聚乙二醇化f多肽及其制备方法与用途 Download PDFInfo
- Publication number
- CN114437182B CN114437182B CN202210111172.0A CN202210111172A CN114437182B CN 114437182 B CN114437182 B CN 114437182B CN 202210111172 A CN202210111172 A CN 202210111172A CN 114437182 B CN114437182 B CN 114437182B
- Authority
- CN
- China
- Prior art keywords
- polypeptide
- pegylated
- peg
- solution
- preparation
- 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.)
- Active
Links
- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 113
- 229920001184 polypeptide Polymers 0.000 title claims abstract description 110
- 102000004196 processed proteins & peptides Human genes 0.000 title claims abstract description 110
- 238000002360 preparation method Methods 0.000 title claims abstract description 10
- 229920001223 polyethylene glycol Polymers 0.000 claims abstract description 31
- 239000002202 Polyethylene glycol Substances 0.000 claims abstract description 15
- 230000002401 inhibitory effect Effects 0.000 claims abstract description 7
- 206010008342 Cervix carcinoma Diseases 0.000 claims abstract description 6
- 208000006105 Uterine Cervical Neoplasms Diseases 0.000 claims abstract description 6
- 201000010881 cervical cancer Diseases 0.000 claims abstract description 6
- 210000004027 cell Anatomy 0.000 claims description 21
- 239000000243 solution Substances 0.000 claims description 17
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 claims description 16
- 238000000034 method Methods 0.000 claims description 12
- 210000004881 tumor cell Anatomy 0.000 claims description 8
- 238000002414 normal-phase solid-phase extraction Methods 0.000 claims description 7
- OIGKWPIMJCPGGD-UHFFFAOYSA-N (2,5-dioxopyrrolidin-1-yl) 3-[2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]ethoxy]propanoate Chemical compound [N-]=[N+]=NCCOCCOCCOCCOCCC(=O)ON1C(=O)CCC1=O OIGKWPIMJCPGGD-UHFFFAOYSA-N 0.000 claims 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 claims description 6
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 6
- 239000003814 drug Substances 0.000 claims description 6
- 230000009435 amidation Effects 0.000 claims description 5
- 238000007112 amidation reaction Methods 0.000 claims description 5
- 238000010612 desalination reaction Methods 0.000 claims description 4
- 239000007853 buffer solution Substances 0.000 claims description 3
- 238000011033 desalting Methods 0.000 claims description 3
- 230000020477 pH reduction Effects 0.000 claims description 3
- 238000010791 quenching Methods 0.000 claims description 3
- 230000000171 quenching effect Effects 0.000 claims description 3
- 239000011541 reaction mixture Substances 0.000 claims description 2
- 238000001179 sorption measurement Methods 0.000 claims description 2
- 125000003277 amino group Chemical group 0.000 abstract description 14
- 230000000694 effects Effects 0.000 abstract description 13
- 230000004048 modification Effects 0.000 abstract description 11
- 238000012986 modification Methods 0.000 abstract description 11
- 150000001540 azides Chemical class 0.000 abstract description 10
- 230000005764 inhibitory process Effects 0.000 abstract description 7
- 230000000975 bioactive effect Effects 0.000 description 15
- 101100505161 Caenorhabditis elegans mel-32 gene Proteins 0.000 description 14
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 9
- 238000006243 chemical reaction Methods 0.000 description 9
- 239000000203 mixture Substances 0.000 description 9
- 239000002904 solvent Substances 0.000 description 8
- 206010028980 Neoplasm Diseases 0.000 description 7
- 210000004899 c-terminal region Anatomy 0.000 description 7
- 239000000047 product Substances 0.000 description 7
- 229940024606 amino acid Drugs 0.000 description 6
- 150000001413 amino acids Chemical class 0.000 description 6
- 150000002500 ions Chemical class 0.000 description 6
- 238000001819 mass spectrum Methods 0.000 description 6
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 6
- 241000699670 Mus sp. Species 0.000 description 5
- 238000002474 experimental method Methods 0.000 description 5
- 238000004128 high performance liquid chromatography Methods 0.000 description 5
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 4
- 238000006664 bond formation reaction Methods 0.000 description 4
- 239000007795 chemical reaction product Substances 0.000 description 4
- 238000004704 ultra performance liquid chromatography Methods 0.000 description 4
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 3
- 238000000134 MTT assay Methods 0.000 description 3
- 231100000002 MTT assay Toxicity 0.000 description 3
- 125000003368 amide group Chemical group 0.000 description 3
- 150000001408 amides Chemical class 0.000 description 3
- 201000011510 cancer Diseases 0.000 description 3
- 235000019253 formic acid Nutrition 0.000 description 3
- 125000000524 functional group Chemical group 0.000 description 3
- 238000000338 in vitro Methods 0.000 description 3
- 230000035755 proliferation Effects 0.000 description 3
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- 239000000427 antigen Substances 0.000 description 2
- 102000036639 antigens Human genes 0.000 description 2
- 108091007433 antigens Proteins 0.000 description 2
- 125000000852 azido group Chemical group *N=[N+]=[N-] 0.000 description 2
- 210000000170 cell membrane Anatomy 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 230000001419 dependent effect Effects 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- WGCNASOHLSPBMP-UHFFFAOYSA-N hydroxyacetaldehyde Natural products OCC=O WGCNASOHLSPBMP-UHFFFAOYSA-N 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- 238000002372 labelling Methods 0.000 description 2
- 230000014759 maintenance of location Effects 0.000 description 2
- 238000004949 mass spectrometry Methods 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 239000007921 spray Substances 0.000 description 2
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 2
- 230000004083 survival effect Effects 0.000 description 2
- 238000001269 time-of-flight mass spectrometry Methods 0.000 description 2
- 238000011282 treatment Methods 0.000 description 2
- 229910021642 ultra pure water Inorganic materials 0.000 description 2
- 239000012498 ultrapure water Substances 0.000 description 2
- PNDPGZBMCMUPRI-HVTJNCQCSA-N 10043-66-0 Chemical compound [131I][131I] PNDPGZBMCMUPRI-HVTJNCQCSA-N 0.000 description 1
- 125000004042 4-aminobutyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])N([H])[H] 0.000 description 1
- 206010006187 Breast cancer Diseases 0.000 description 1
- 208000026310 Breast neoplasm Diseases 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- 238000012404 In vitro experiment Methods 0.000 description 1
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 description 1
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 1
- 239000004472 Lysine Substances 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- 208000024770 Thyroid neoplasm Diseases 0.000 description 1
- ATJFFYVFTNAWJD-UHFFFAOYSA-N Tin Chemical compound [Sn] ATJFFYVFTNAWJD-UHFFFAOYSA-N 0.000 description 1
- 229920004890 Triton X-100 Polymers 0.000 description 1
- 239000013504 Triton X-100 Substances 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- 239000002671 adjuvant Substances 0.000 description 1
- 230000001093 anti-cancer Effects 0.000 description 1
- 230000005775 apoptotic pathway Effects 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 229940022399 cancer vaccine Drugs 0.000 description 1
- 238000009566 cancer vaccine Methods 0.000 description 1
- 229910002092 carbon dioxide Inorganic materials 0.000 description 1
- 239000001569 carbon dioxide Substances 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 230000003833 cell viability Effects 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 239000003599 detergent Substances 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 150000002148 esters Chemical class 0.000 description 1
- 238000005194 fractionation Methods 0.000 description 1
- 230000024924 glomerular filtration Effects 0.000 description 1
- 125000003827 glycol group Chemical group 0.000 description 1
- VKYKSIONXSXAKP-UHFFFAOYSA-N hexamethylenetetramine Chemical compound C1N(C2)CN3CN1CN2C3 VKYKSIONXSXAKP-UHFFFAOYSA-N 0.000 description 1
- 230000005745 host immune response Effects 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- ZCYVEMRRCGMTRW-YPZZEJLDSA-N iodine-125 Chemical compound [125I] ZCYVEMRRCGMTRW-YPZZEJLDSA-N 0.000 description 1
- 229940044173 iodine-125 Drugs 0.000 description 1
- 229960000310 isoleucine Drugs 0.000 description 1
- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 description 1
- 238000004811 liquid chromatography Methods 0.000 description 1
- 238000001294 liquid chromatography-tandem mass spectrometry Methods 0.000 description 1
- -1 lys11 Proteins 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- DEQXHPXOGUSHDX-UHFFFAOYSA-N methylaminomethanetriol;hydrochloride Chemical compound Cl.CNC(O)(O)O DEQXHPXOGUSHDX-UHFFFAOYSA-N 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 230000006320 pegylation Effects 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 238000012510 peptide mapping method Methods 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000000770 proinflammatory effect Effects 0.000 description 1
- 230000009257 reactivity Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000004007 reversed phase HPLC Methods 0.000 description 1
- 230000019491 signal transduction Effects 0.000 description 1
- 238000010532 solid phase synthesis reaction Methods 0.000 description 1
- 239000011550 stock solution Substances 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000001308 synthesis method Methods 0.000 description 1
- 238000004885 tandem mass spectrometry Methods 0.000 description 1
- 201000002510 thyroid cancer Diseases 0.000 description 1
- 239000011573 trace mineral Substances 0.000 description 1
- 235000013619 trace mineral Nutrition 0.000 description 1
- 238000001195 ultra high performance liquid chromatography Methods 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
- 238000005303 weighing Methods 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
- C07K14/001—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof by chemical synthesis
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
- A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
- A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
- C07K1/1072—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups
- C07K1/1077—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups by covalent attachment of residues other than amino acids or peptide residues, e.g. sugars, polyols, fatty acids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Medicinal Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Pharmacology & Pharmacy (AREA)
- Biophysics (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Genetics & Genomics (AREA)
- Animal Behavior & Ethology (AREA)
- Engineering & Computer Science (AREA)
- Epidemiology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Analytical Chemistry (AREA)
- Gastroenterology & Hepatology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Peptides Or Proteins (AREA)
Abstract
本申请公开了一种聚乙二醇化F多肽及其制备方法与用途。本发明的技术方案是在F多肽上进行聚乙二醇化修饰，即在F多肽的氨基上修饰叠氮化四聚乙二醇，使得该聚乙二醇化F多肽具有显著的人宫颈癌HeLa细胞抑制活性。其中，叠氮四聚乙二醇化F1多肽较修饰前F1多肽对于HeLa细胞的抑制活性提高了约3.7倍；叠氮四聚乙二醇化F3多肽较修饰前F3多肽对于HeLa细胞的抑制活性提高了约6倍。
Description
技术领域
本发明生物多肽领域，涉及一种聚乙二醇化F多肽，尤其涉及一种聚乙二醇化F多肽及其制备方法与用途，该聚乙二醇化F多肽具有显著的人宫颈癌HeLa细胞抑制活性。
背景技术
研究发现F1、F3多肽在体外对多种癌细胞系的增值具有明显的抑制作用，包括TC-1，HeLa及B16(见专利PCT/CN2017/000744，201811161509.9)。体外试验成功后，又在体内试验了F多肽，以检测其是否能引起荷瘤小鼠的宿主免疫反应，从而作为癌症疫苗的佐剂。使用单独或与E6/7抗原组合的F多肽的肿瘤局部注射对腹腔内植入TC-1细胞的小鼠进行了测试。与仅使用肽或对照组相比，F多肽与E6/7抗原的组合显着提高了小鼠的存活率。当针对TC-1细胞进行测试时，F多肽能够在体内激活TC-1肿瘤细胞上的NFκB信号通路。通过该途径释放更多促炎细胞因子，然后渗透到肿瘤微环境并导致癌细胞凋亡。除TC-1细胞外，F2和F3多肽很大程度上抑制宫颈癌HeLa细胞系体外增殖，且具有累加抑制作用，该作用是通过调节TNFα依赖性细胞凋亡途径来进行的。我们也开展了关于F多肽的结构修饰以提高抗癌活性的研究。将F多肽与碘125或131放射性同位素结合以治疗乳腺癌和未分化甲状腺癌。小鼠试验表明，与非标记治疗相比，用这些药物标记F多肽显着提高了荷瘤小鼠的存活率(见专利CN202010210509.4，ZL201711167995.0)。
大量文献显示，聚乙二醇化的多肽的主要技术效果包括可以增加多肽的稳定性从而增加其半衰期，同时提高溶解度。由于聚乙二醇有较强亲水性，F多肽与聚乙二醇分子的结合会提高它们的整体亲水性，从而提高溶解度，意味着最终可降低肾小球滤过。发生这种情况的机制是由于氨基转化为酰胺以及已有酰胺连接聚乙二醇结构，增加修饰后分子的正电荷。具有较高离子电荷的分子往往被肾脏超滤清除得更慢，因此会延长药物在血液中的半衰期。然而，目前尚未有聚乙二醇修饰可增加多肽生物抑制活性的报道。
发明内容
为解决现有技术的缺陷，本发明对F多肽进行了聚乙二醇化修饰，即在F1多肽和F3多肽上修饰叠氮四聚乙二醇。该方法通过使用叠氮四聚乙二醇化加强体外F多肽对宫颈癌HeLa细胞的作用。
为解决上述目的，本发明一方面公开了一种聚乙二醇化F多肽，包含一种或多种R1修饰的F多肽；所述F多肽为F1多肽或F3多肽，所述F1多肽或F3多肽的C端酰胺化；
所述F1多肽或F3多肽上任选地连接有1-3个R1基团：叠氮四聚乙二醇，其结构如下所示，
所述F1多肽的序列如SEQ ID NO.1所示；
所述F3多肽的序列如SEQ ID NO.2所示；SEQ ID NO.1：GLLSVLGSVAKHVLPHVLPHVVPVIAEHL-NH2；
SEQ ID NO.2：GLFGVLGSIAKHVLPHVVPVIAEKL-NH2。
作为优选，所述R1基团连接于F多肽的氨基。
作为优选，所述F多肽的氨基是指F多肽中未参与形成肽键的氨基以及C端酰胺化所得氨基。
作为优选，所述F多肽的氨基是指甘氨酸、赖氨酸中未参与形成肽键的氨基以及异亮氨酸C端酰胺化所得氨基。
作为优选：
所述F1多肽中Glyl、Lys11未参与形成肽键的氨基以及Leu29的C端酰胺化氨基中任选的1-3个氨基上连有R1基团；
所述F3多肽中Gly1、Lys11、Lys24未参与形成肽键的氨基以及Leu25的C端酰胺化氨基中任选的1-3个氨基上连有R1基团。
具体地，所述的聚乙二醇化F多肽包含一种或多种具有式1-12所示结构的多肽：
本发明另一方面还提供了一种上述聚乙二醇化F多肽的制备方法，其特征在于，包括以下步骤：
1)将NHS-PEG4-Azide溶解于二甲基亚砜中得到PEG溶液；
2)将F1或F3多肽溶解于PBS中，制备成F1或F3多肽溶液；
3)将步骤2)制备的多肽溶液与步骤1)中的PEG溶液以1：20的摩尔比例充分混合，并在室温下反应1h；
4)然后加入1M Tris-HCl缓冲液进行淬灭，至Tris-HCl终浓度为50-100mM，在室温下孵育反应5分钟；
5)向步骤4)的反应混合物加入10％三氟乙酸进行酸化，至三氟乙酸浓度为0.1％，然后脱盐经固相萃取柱纯化得到所述聚乙二醇化F多肽。
作为优选，所述步骤1)PEG溶液中NHS-PEG4-Azide的浓度为250mM；所述步骤2)多肽溶液中F1或F3多肽的浓度为5mg/mL。
作为优选，所述步骤5)脱盐所用的方法是固相萃取柱吸附脱盐，所用的固相萃取柱型号为Sep-Pak C18。
所述肿瘤细胞包括但不限于HPV阳性细胞、HPV阴性细胞，优选人宫颈癌HeLa细胞。
本发明具有以下有益效果：
聚乙二醇修饰(叠氮四聚乙二醇)的F多肽相较于未修饰的F多肽具有更强的HeLa细胞抑制活性。叠氮四聚乙二醇化F1多肽较修饰前F1多肽对于HeLa细胞的抑制活性提高了约3.7倍；叠氮四聚乙二醇化F3多肽较修饰前F3多肽对于HeLa细胞的抑制活性提高了约6倍。此显著作用主要是通过叠氮四聚乙二醇化修饰增加原F多肽的正电荷，进而与癌细胞膜有更强的相互作用来影响细胞膜及基质结构。
附图说明
图1为NHS-PEG4-Azide与F1多肽反应产物的超高效液相色谱实验结果；
图2为NHS-PEG4-Azide与F3多肽反应产物的超高效液相色谱实验结果；
图3为活性馏分bioactive PEG-F1所包含的聚乙二醇化F1多肽的结构式；
图4为活性馏分bioactive PEG-F3-a所包含的聚乙二醇化F3多肽的结构式；
图5为活性馏分bioactive PEG-F3-b所包含的聚乙二醇化F3多肽的结构式；
图6为MTT实验结果。
具体实施方式
以下将通过实施例来详细说明本申请的实施方式，借此对本申请如何应用技术手段来解决技术问题并达成技术功效的实现过程能充分理解并据以实施。
本申请中所用原料、设备，若无特别说明，均为本领域的常用原料、设备，均来自市售产品。本申请中所用方法，若无特别说明，均为本领域的常规方法。
本申请还存在其它多种可实施的技术方案，在此不做一一列举，本申请权利要求中要求保护的技术方案都是可以实施的。
“包含”或“包括”旨在表示组合物(例如介质)和方法包括所列举的要素，但不排除其他要素。当用于定义组合物和方法时，“基本上由......组成”意味着排除对于所述目的的组合具有任何重要意义的其他要素。因此，基本上由本文定义的元素组成的组合物不排除不会实质上影响要求保护的本申请的基本和新颖特征的其他材料或步骤。“由......组成”是指排除其他组成部分的微量元素和实质性的方法步骤。由这些过渡术语中的每一个定义的实施方案都在本申请的范围内。的专利中公开的制备方法获得的。
实验试剂：
F1多肽及F3多肽分别如SEQ ID NO.1及SEQ ID NO.2所示，且其C端被酰胺化。
F1多肽：
SEQ ID NO.1：GLLSVLGSVAKHVLPHVLPHVVPVIAEHL-NH2(C端酰胺化)；
F3多肽：
SEQ ID NO.2：GLFGVLGSIAKHVLPHVVPVIAEKL-NH2(C端酰胺化)。
F1多肽与F3由中国无锡迈拓生物有限公司应用固相合成法合成，并通过反相HPLC确定多肽的纯度＞99％。
NHS-PEG4-Azide：叠氮四聚乙二醇活性酯，化学式为：
实施例1：聚乙二醇化F多肽的合成方法
因为F多肽序列上具有多个酰胺基团，选用了具有酰胺基团反应活性的NHS-PEG4-Azide分子进行F多肽的聚乙二醇化反应，具体步骤如下：
1)将一定量(1mg)的NHS-PEG4-Azide溶解于二甲基亚砜(20μL)中，制备成250mM的原液；
2)将5mg的F1或F3多肽溶解于PBS中，制备成5mg/mL的溶液；
3)将2)制备的多肽溶液与1)中的PEG溶液以1：20的摩尔比例充分混合，然后混合物在室温下反应1小时；
4)然后加入1M Tris-HCl(三羟甲基氨基甲烷盐酸盐)缓冲液进行淬灭，至Tris-HCl终浓度为50-100mM，在室温下孵育反应5分钟；
5)向4)的产物混合物加入10％三氟乙酸进行酸化，至三氟乙酸浓度为0.1％，然后使用Sep-Pak C18固相萃取柱进行脱盐；
6)将5)所得洗脱混合物使用离心冻干机(Thermo ScientificTM SavantTMSpeedVac)干燥并储存在-80℃的冰箱中以用于以下高效液相色谱分离纯化。
实施例2：高效液相色谱纯化分离
将聚乙二醇化的F多肽在超高效液相色谱(Agilent 1290Infinity II)上进行连续分馏。所用色谱柱为AdvanceBio Peptide Mapping 2.1x150mm，2.7μm。使用两种溶剂：0.1％三氟乙酸的超纯水溶液(A)和0.1％三氟乙酸的100％乙腈溶液(B)，流速为0.4mL/min，色谱信号波长为210nm。具体线性梯度如下表1和2所示。将收集到的馏分(见图1、2)用SpeedVac真空浓缩器(Thermofisher Scientific)冻干，并于-20度存储待用。
表1高效液相色谱分离聚乙二醇化的F1的方法
| Time(min) | A(％) | B(％) |
| 0.00 | 97.00 | 3.00 |
| 2.00 | 97.00 | 3.00 |
| 4.00 | 34.40 | 65.60 |
| 7.75 | 32.30 | 67.70 |
| 14.00 | 32.21 | 67.79 |
表2高效液相色谱分离聚乙二醇化的F3的方法
| Time(min) | A(％) | B(％) |
| 0.00 | 97.00 | 3.00 |
| 2.00 | 97.00 | 3.00 |
| 5.00 | 33.30 | 66.70 |
| 20.00 | 33.24 | 66.76 |
如图1所示即为NHS-PEG4-Azide与F1多肽反应产物的超高效液相色谱实验结果。其中，活性馏分bioactive PEG-F1被标出。活性馏分bioactive PEG-F1的保留时间范围为11.4至12.8min，峰值在12.059min，占所有色谱峰总面积的12.5148％。此馏分被收集并冻干。
如图2所示即为NHS-PEG4-Azide与F3多肽反应产物的超高效液相色谱实验结果。其中，活性馏分bioactive PEG-F3-a和bioactive PEG-F3-b被标出。活性馏分bioactivePEG-F3-a和bioactive PEG-F3-b的保留时间范围分别为15.6至17.5min、17.75至20min，峰值分别在16.391和18.609min，占所有色谱峰总面积分别为7.5744％和12.3378％。此两个馏分被分别收集并冻干。
实施例3：产物结构表征
冻干的活性馏分被重新溶解于50μL的0.1％的甲酸+2％乙腈+98％超纯水的混合溶液中制成0.1μg/μL的多肽溶液，并用四极杆飞行时间质谱(SCIEX，Concord，加拿大)进行分析。将样品在与QTOF X500R质谱仪(SCIEX，Concord，加拿大)耦合的ExionLC液相色谱系统上通过LC-MS/MS进行分析)配备电喷雾离子源。将15μL的样品注射到配备有用于质谱分析的SecurityGuard色谱柱的100mm×1.7μm Aeris PEPTIDE XB-C18100uHPLC色谱柱(Phenomenex，悉尼，澳大利亚)上。5-35％溶剂B的线性梯度超过10min，流速为400μL/min，然后是更陡峭的梯度，在2min内从35％到80％溶剂B和在1分钟内从80％到95％溶剂B用于肽洗脱。溶剂B在95％的浓度下保持1分钟以清洗色谱柱，然后在下一次进样之前返回到5％的溶剂B进行平衡。溶剂A由0.1％甲酸(aq)组成，溶剂B包含100％乙腈/0.1％甲酸(aq)。离子喷雾电压设置为5500V，去簇电位(DP)100V，帘幕气流30，离子源气体1(GS1)40，离子源气体2(GS2)50，喷雾温度为450℃。质谱仪以信息相关采集、IDA模式采集质谱数据。全扫描TOFMS数据在质量范围350-1400和子离子ms/ms50-1800内采集。在TOF-MS扫描中观察到的离子超过100cps的阈值和+2到+5的电荷状态被设置为触发产物离子的采集。使用SCIEX OS软件获取和处理数据。MS/MS结果如表3所示，活性馏分为聚乙二醇化多肽的混合物，其中检测到了NHS-PEG4-Azide与F1或F3多肽两端的酰胺基及序列中赖氨酸支链上的酰胺中一个或多个反应的产物。因此，目前我们认为该馏分的高活性是由混合物引起的。
表3
/>
图3所示即为活性馏分bioactive PEG-F1所包含的聚乙二醇化F1多肽的结构式，根据质谱结果其具体包含5种结构，分别为式1、式2、式3、式4、式5。质谱结果(表3)显示出活性馏分bioactive PEG-F1中NHS-PEG4-Azide可以与F1多肽(SEQ ID NO.1)中的氨基酸Gly1，Lys11以及C端的酰胺化的Leu29中的一个或多个的-NH2官能团反应，检测到的参与反应的独立氨基酸及组合包括Gly1，Lys11，Gly1+Lys11，Gly1+Leu29，Gly1+Lys11+Leu29。
图4所示即为活性馏分bioactive PEG-F3-a所包含的聚乙二醇化F3多肽的结构式，根据质谱结果其具体包含6种结构，分别为式6、式7、式8、式9、式10、式11。质谱结果(表3)显示出活性馏分bioactive PEG-F3-a中NHS-PEG4-Azide可以与F3多肽(SEQ ID NO.2)中的氨基酸Gly1，Lys11，Lys24以及C端的酰胺化的Leu25中的一个或多个的-NH2官能团反应，检测到的参与反应的独立氨基酸及组合包括Gly1，Lys11，Leu25，Gly1+Lys11，Gly1+Leu25，Gly1+Lys11+Lys24。
图5所示即为活性馏分bioactive PEG-F3-b所包含的聚乙二醇化F3多肽的结构式，根据质谱结果其具体包含3种结构，分别为式8、式10、式11。质谱结果(表3)显示出活性馏分bioactive PEG-F3-b中NHS-PEG4-Azide可以与F3多肽(SEQ ID NO.2)中的氨基酸Gly1，Lysl1以及C端的酰胺化的Leu25中的一个或多个的-NH2官能团反应，检测到的参与反应的独立氨基酸及组合包括Leu25，Gly1+Leu25，Gly1+Lys11+Leu25。实施例4：HeLa细胞活力和增殖的MTT检测
HeLa细胞系在完全培养基中培养，并补充有10％热灭活FBS、100U青霉素/mL和100μg链霉素。培养物将维持在37℃和5％CO2培养过夜。应用MTT测定验证了馏分的活性：HeLa细胞在平底96孔板中单独培养。将F1、F3以及高效液相色谱收集的活性聚乙二醇化多肽组分(包括PEG-F1、PEG-F3-a和PEG-F3-b)10μg/mL的浓度加入1×104个/100uL HeLa细胞中，并在37℃下培养过夜，5％二氧化碳。用Triton X-100(Anitha et al.，2012)和31nMcaerin 1.1处理的孔用作阳性对照。每个处理一式三份进行。加入10微升MTT试剂，再培养2小时，然后加入100毫升去污剂，室温避光放置2小时。根据制造商的方案，使用微量滴定板读数器(BioTek，美国)在570nm处分析结果。图6显示相同浓度下，PEG-F1，PEG-F3-a和PEG-F3-b显著提高了F1和F3对于HeLa细胞的增殖。
本申请说明书中未作详细描述的内容属于本领域技术人员的公知常识。
如在通篇说明书及权利要求当中所提及的“包含”为一开放式用语故应解释成“包含但不限定于”。“大致”是指在可接收的误差范围内，本领域技术人员能够在一定误差范围内解决所述技术问题，基本达到所述技术效果。
还需要说明的是，术语“包括”、“包含”或者其任何其他变体意在涵盖非排他性的包含，从而使得包括一系列要素的商品或者系统不仅包括那些要素，而且还包括没有明确列出的其他要素，或者是还包括为这种商品或者系统所固有的要素。在没有更多限制的情况下，由语句“包括一个......”限定的要素，并不排除在包括所述要素的商品或者系统中还存在另外的相同要素。
上述说明示出并描述了本申请的若干优选实施例，但如前所述，应当理解本申请并非局限于本文所披露的形式，不应看作是对其他实施例的排除，而可用于各种其他组合、修改和环境，并能够在本文所述发明构想范围内，通过上述教导或相关领域的技术或知识进行改动。而本领域人员所进行的改动和变化不脱离本申请的精神和范围，则都应在本申请所附权利要求的保护范围。
Claims (4)
1.一种聚乙二醇化F多肽在制备具有肿瘤细胞抑制活性的药物中的用途，其特征在于，所述肿瘤细胞包括HPV阳性细胞和HPV阴性细胞，所述聚乙二醇化F多肽的制备方法按如下步骤进行：
1）将NHS-PEG4-Azide溶解于二甲基亚砜中得到PEG溶液；
2）将F1或F3多肽溶解于PBS中，制备成F1或F3多肽溶液；
3）将步骤2）制备的多肽溶液与步骤1）中的PEG溶液以1：20的摩尔比例充分混合，并在室温下反应1 h；
4）然后加入1M Tris-HCl 缓冲液进行淬灭，至Tris-HCl终浓度为 50-100mM，在室温下孵育反应 5 分钟；
5）向步骤4）的反应混合物加入10%三氟乙酸进行酸化，至三氟乙酸浓度为0.1%，然后脱盐经固相萃取柱纯化得到所述聚乙二醇化F多肽；
所述F1多肽或F3多肽的C端酰胺化；
所述F1多肽的序列如SEQ ID NO. 1所示；
所述F3多肽的序列如SEQ ID NO. 2所示；
SEQ ID NO. 1: GLLSVLGSVAKHVLPHVLPHVVPVIAEHL-NH2；
SEQ ID NO. 2: GLFGVLGSIAKHVLPHVVPVIAEKL-NH2。
2.根据权利要求1所述的聚乙二醇化F多肽在制备具有肿瘤细胞抑制活性的药物中的用途，其特征在于：所述肿瘤细胞为人宫颈癌HeLa细胞。
3.根据权利要求1所述的聚乙二醇化F多肽在制备具有肿瘤细胞抑制活性的药物中的用途，其特征在于：所述步骤1）PEG溶液中NHS-PEG4-Azide的浓度为250mM；所述步骤2）多肽溶液中F1或F3多肽的浓度为5mg/mL。
4.根据权利要求1所述的聚乙二醇化F多肽在制备具有肿瘤细胞抑制活性的药物中的用途，其特征在于：所述步骤5）脱盐所用的方法是固相萃取柱吸附脱盐，所用的固相萃取柱型号为Sep-Pak C18。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210111172.0A CN114437182B (zh) | 2022-01-29 | 2022-01-29 | 聚乙二醇化f多肽及其制备方法与用途 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210111172.0A CN114437182B (zh) | 2022-01-29 | 2022-01-29 | 聚乙二醇化f多肽及其制备方法与用途 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN114437182A CN114437182A (zh) | 2022-05-06 |
| CN114437182B true CN114437182B (zh) | 2024-02-23 |
Family
ID=81370876
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210111172.0A Active CN114437182B (zh) | 2022-01-29 | 2022-01-29 | 聚乙二醇化f多肽及其制备方法与用途 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114437182B (zh) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115957306B (zh) * | 2022-11-22 | 2023-08-22 | 中奥生物医药技术(广东)有限公司 | 一种Caerin1.1/1.9联合抗CD47抗体在制备治疗黑色素瘤药物中的应用 |
| CN116789769B (zh) * | 2023-07-31 | 2024-02-23 | 中奥生物医药技术(广东)有限公司 | 一种f3多肽的制备方法 |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102827270A (zh) * | 2012-09-13 | 2012-12-19 | 无锡和邦生物科技有限公司 | 一种聚乙二醇化艾塞那肽衍生物及其用途 |
| CN109200270A (zh) * | 2018-09-30 | 2019-01-15 | 倪国颖 | 一种能提高多肽疫苗对hpv感染肿瘤治疗效果的联合用药物及其应用 |
-
2022
- 2022-01-29 CN CN202210111172.0A patent/CN114437182B/zh active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102827270A (zh) * | 2012-09-13 | 2012-12-19 | 无锡和邦生物科技有限公司 | 一种聚乙二醇化艾塞那肽衍生物及其用途 |
| CN109200270A (zh) * | 2018-09-30 | 2019-01-15 | 倪国颖 | 一种能提高多肽疫苗对hpv感染肿瘤治疗效果的联合用药物及其应用 |
Non-Patent Citations (2)
| Title |
|---|
| Synthesized natural peptides from amphibian skin secretions increase the efficacy of a therapeutic vaccine by recruiting more T cells to the tumour site;Xuan Pan等;《BMC Complementary and Alternative Medicine》;第19卷(第1期);第2 页右栏第1-2段 * |
| 氨基叠氮聚乙二醇的制备;刘 佳等;《精细化工》;第36卷(第8期);摘要，全文 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN114437182A (zh) | 2022-05-06 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN114437182B (zh) | 聚乙二醇化f多肽及其制备方法与用途 | |
| KR102302392B1 (ko) | 항염증 활성을 갖는 펩티드 및 이를 포함하는 조성물 | |
| EP2213680B1 (en) | Peptides having pharmacological activity for treating disorders associated with altered cell migration, such as cancer | |
| AU2019203671B2 (en) | Cyclotides as immunosuppressive agents | |
| US20060264605A1 (en) | Polypeptides having anti-HIV activity and compositions comprising same | |
| KR101310511B1 (ko) | 흉선-특이성 단백질 | |
| MX2013004350A (es) | Analogos de peptido insulinotropico dependientes de glucosa. | |
| Wittmann-Liebold | Studies on the primary structure of 20 proteins from Escherichia coli ribosomes by means of an improved protein sequenator | |
| CA2068327C (en) | Polypeptide and anti-hiv drug prepared therefrom | |
| Youn et al. | High-yield production of biologically active mono-PEGylated salmon calcitonin by site-specific PEGylation | |
| Brown et al. | Affinity purification of synthetic peptides and proteins on porous graphitised carbon | |
| Ibrahim et al. | Callyaerin G, a new cytotoxic cyclic peptide from the marine sponge Callyspongia aerizusa | |
| Gesner-Apter et al. | Three novel metabolites from a bloom of the cyanobacterium Microcystis sp. | |
| RU2669939C2 (ru) | Трипептиды | |
| Nabika et al. | Synthesis of IB-01212 by multiple N-methylations of peptide bonds | |
| CN114716509B (zh) | 一种兼具抗氧化和降血压作用的活性肽及其应用 | |
| CN114736269B (zh) | 一种客家黄酒抗氧化和降血压活性肽及其应用 | |
| WITTMANN-LIEBOLD | Ribosomal Proteins, XXI, Amino Acid Composition of the Tryptic Peptides isolated from Proteins S4, S18 and S20 of Eschenchia coli Ribosomes | |
| Roy et al. | Barangamide A, a new cyclic peptide from the Indonesian sponge Theonella swinhoei | |
| KR102193211B1 (ko) | 비오틴 모이어티가 결합된 폴리펩티드 및 이를 포함하는 경구 투여용 약학적 조성물 | |
| CN110551179B (zh) | 一种经修饰的抗hiv多肽及其制备方法和用途 | |
| CN109553658B (zh) | Ang-(1-7)的氮杂多肽类似物、制备方法及应用 | |
| EP3398959B1 (en) | Method for preparing lixisenatide | |
| US20120238491A1 (en) | Immunologically active lipopeptides and methods for use thereof | |
| CN114456237B (zh) | 海绵环肽类化合物及其制备方法与应用 |
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 | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |