CN105315348A - Transmembrane polypeptide, preparation method and application thereof - Google Patents
Transmembrane polypeptide, preparation method and application thereof Download PDFInfo
- Publication number
- CN105315348A CN105315348A CN201410281405.7A CN201410281405A CN105315348A CN 105315348 A CN105315348 A CN 105315348A CN 201410281405 A CN201410281405 A CN 201410281405A CN 105315348 A CN105315348 A CN 105315348A
- Authority
- CN
- China
- Prior art keywords
- polypeptide
- cell
- conjugate
- present
- synthesis
- 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.)
- Granted
Links
Landscapes
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention belongs to the technical field of medicines and provides a transmembrane polypeptide which is composed of 13 amino acids with the sequence being RGGRVRRRWRGGR. The polypeptide is prepared through solid phase synthesis and then is coupled to an anti-tumor drug through a chemical synthesis method. The conjugate provided in the invention has the effects of passing through blood brain barrier and inhibiting tumors.
Description
Technical field
The invention belongs to medical art, relate to the pharmaceutical field that tumour is relevant, specifically, the present invention relates to a kind of transmembrane polypeptide, and the conjugate that this polypeptide and antitumour drug coupling are formed.
Background technology
The sickness rate of brain tumor and metastatic encephaloma rises year by year.To in its treatment, hemato encephalic barrier is considered to the major cause stoping chemotherapeutics to enter cerebral tissue and then affect the treatment.Hemato encephalic barrier is that body participates in one of internal barrier of inherent immunity, be made up of the brain capillary wall of the pia mater between circulation of blood and brain essence, choroid plexus and the cutose be wrapped in outside wall, can stopped that causal organism and other macromolecular substance enter cerebral tissue and the ventricles of the brain by circulation of blood.Substantially the macromolecular drug of 100%, comprises polypeptide, recombinant protein, monoclonal antibody, all cannot pass through hemato encephalic barrier based on the medicine, gene therapy related drugs etc. of RNA perturbation technique, and the small-molecule drug being greater than 98% also cannot pass hemato encephalic barrier.At present, radiation treatment and chemotherapy can only extend patient vitals 4 months, and therefore the present invention is devoted to research and makes existing medicine better through hemato encephalic barrier.
Polypeptide has polar terminals and non-polar end due to it, therefore has higher availability in vivo, due to itself be endogenous material so toxicity is little, but polypeptide is directly subject to larger restriction as medicine, such as its poor stability, can not be oral.In recent years, polypeptide is as application one of focus becoming new drug development gradually of medicine subsidiary function.Cell-penetrating peptide is the peptide molecule of the class tool specific function starting to recognize mid-term in 20th century, and English academic title is generally written as cell2-penetrating-peptides (CPPs).They are made up of 5 ~ 30 amino acid usually, and typically general have membranes penetration function by 8 ~ 16 Amino acid profiles, also can carry other molecule even supramolecule particle enter in cell.Exactly because this special function, they are counted as the effective intracellular transport instrument of bioactive molecules, are with a wide range of applications.
Polypeptide and medicine formed the example of composition much by covalent manner in the last few years, such as: polypeptide Penetratin and the coupling of Zorubicin covalency are used for the treatment of mammary cancer; Peptide T AT and the coupling of medicine P53 covalency are used for the treatment of cancer eye transfer; Polypeptide YTA4 is coupled with methotrexate covalency and treats mammary cancer.
Summary of the invention
Below detailed description of the present invention:
(1) of the present inventionly film peptide is worn
Cell-penetrating peptide of the present invention, is made up of 13 amino acid, and is wetting ability and hydrophobicity alternate combinations, and aminoacid sequence is:
RGGRVRRRWRGGR
This polypeptide itself does not have physiologically active, but has the effect through hemato encephalic barrier, the invention still further relates to preparation method and the application thereof of this polypeptide.
(2) preparation method wearing film peptide of the present invention
Polypeptide required for the present invention adopts the method for solid phase to prepare, and solid phase synthesis take solid support resin as carrier, adds amino acid whose process from C end and carboxyl terminal gradually to N end and aminoterminal.
Preferred resin in the present invention: Fmoc-wang resin, 2-cl-trt resin, resin substitution value 0.24 ~ 0.4.
Protected amino acid used in the present invention is: Fmoc-Arg (pbf)-OH, Fmoc-Gly-OH, Fmoc-Val-OH, Fmoc-Trp-OH, and in reaction process, protected amino acid 3 ~ 5 times is excessive.
Deprotecting regent used in the present invention is: piperidines/DMF, and ratio is 10:90 ~ 30:70.
Coupling reagent used in the present invention is: 1. benzotriazole-N, N, N', N'-tetramethyl-urea hexafluorophosphate/I-hydroxybenzotriazole (HOBT), 2. 2-(7-azo benzotriazole)-N, N, N', N'-tetramethyl-urea phosphofluoric acid ester/I-hydroxybenzotriazole (HOBT), 3. N, N'-DIC/I-hydroxybenzotriazole (HOBT).
1. or the 2. plant coupling reagent in the present invention, if use the, so acid binding agent should be added in process in reaction: DIEA/NMP.
The cutting reagent used in the present invention is: TFA/ thioanisole/water/phenol/1，2-ethandithiol, and ratio is: 82.5:5:5:5; 2.5.
In the present invention use MS-IT-TOF to determine molecular weight, use HPLC purification of crude peptide
(3) method of attachment of polypeptide of the present invention and antitumor drug Zorubicin
1) synthesis of Compound II per
1.0g (1.72mmol) Zorubicin is added, 0.52g (5.20mmol) Succinic anhydried, 30mlTHF in 50m reaction flask, drip 0.66g (5.12mmol) N, N-diisopropylethylamine (DIEA), 27 DEG C are stirred 6h, and solvent evaporated obtains wax, add water stirring, there is yellow solid to separate out, suction filtration, wash twice, drying, obtains yellow solid 1.15g.
2) synthesis of Compound I
0.22g (0.32mmol) Compound II per is added, 0.10g (0.31mmol) O-benzotriazole-N, N in 50m reaction flask; N'; N'-tetramethyl-urea Tetrafluoroboric acid ester (TBTU), 5mlDMF, nitrogen protection; drip 0.08g (0.62mmol) N; N-diisopropylethylamine (DIEA), after 27 DEG C of stirring 1h, adds 0.10g polypeptide; after 27 DEG C of stirring 3h, termination reaction.Use MS-IT-TOF to determine molecular weight, use HPLC purification of crude product.
(4) method of attachment of polypeptide of the present invention and antitumor drug paclitaxel
1) synthesis of compound III
1.50g (1.72mmol) taxol is added, 0.52g (5.20mmol) Succinic anhydried, 30mlTHF in 50m reaction flask, drip 0.66g (5.12mmol) N, N-diisopropylethylamine (DIEA), 27 DEG C are stirred 6h, and solvent evaporated obtains wax, add water stirring, there is yellow solid to separate out, suction filtration, wash twice, drying, obtains yellow solid 1.15g.
2) synthesis of compound IV
0.22g (0.32mmol) compound III is added, 0.10g (0.31mmol) O-benzotriazole-N, N in 50m reaction flask; N'; N'-tetramethyl-urea Tetrafluoroboric acid ester (TBTU), 5mlDMF, nitrogen protection; drip 0.08g (0.62mmol) N; N-diisopropylethylamine (DIEA), after 27 DEG C of stirring 1h, adds 0.10g polypeptide; after 27 DEG C of stirring 3h, termination reaction.Use MS-IT-TOF to determine molecular weight, use HPLC purification of crude product.
Accompanying drawing explanation
Fig. 1 is the HPLC test result figure of improvement on synthesis.
Embodiment
Below in conjunction with embodiment, the present invention is further described in detail, the embodiment provided only in order to illustrate the present invention, instead of in order to limit the scope of the invention.
Below in an example, the various process do not described in detail and method are ordinary methods as known in the art.
embodiment 1: the solid phase synthesis of polypeptide
Use the solid-phase peptide synthesis (amino acid of use is purchased from Shanghai gill company) of Fmoc strategy, the Liberitity type instrument using CEM company to produce carries out the synthesis of transmembrane polypeptide of the present invention.Working method is carried out according to the instrument specification sheets of manufacturer.
Synthesis agents useful for same is selected:
(1) vector resin: Fmoc-Arg (pbf)-wang-rink, substitution degree: 0.33
(2) protected amino acid selected by: Fmoc-Arg (pbf)-OH, Fmoc-Gly-OH, Fmoc-Val-OH, Fmoc-Trp-OH, in reaction, protected amino acid used 5 times is excessive.
(3) deprotecting regent used in the present invention is: piperidines/DMF, and ratio is 20:80.
(4) coupling reagent used in the present invention is: N, N'-DIC (DIC)/I-hydroxybenzotriazole (HOBT).
(5) cutting reagent used in the present invention is: TFA/ thioanisole/water/phenol/1，2-ethandithiol, and ratio is: 82.5:5:5:5; 2.5.
HPLCC18 semipreparative column (purchased from Phenomenex company) is used to carry out purifying obtained polypeptide, testing conditions is: determined wavelength: 214nm, mobile phase A: acetonitrile (containing 0.1% trifluoroacetic acid), Mobile phase B: water (containing 0.1% trifluoroacetic acid), elution requirement: by 35%A ~ 50%A in 30 minutes, MS-IT-TOF is utilized to determine gained sterling molecular weight, determine sample purity with HPLC, purity is greater than 95% and obtains polypeptides freeze-dry powder through desalination and lyophilize.
HPLC test result is shown in Fig. 1, table 1.
Table 1HPLC test result
embodiment 2: conjugate is through the interior evaluating of hemato encephalic barrier
By above-mentioned conjugate I and contrast Zorubicin, conjugate IV and contrast taxol, 10mg/ml solution is configured to physiological saline, by rat (purchased from Chinese Academy of Sciences's Shanghai Experimental Animal Center, SD rat) with etherization, through vena femoralis injection (200 μ L/, 6/group), within latter 30 minutes, get cerebrospinal fluid respectively at injection, and disconnected neck puts to death rat.Cerebrospinal fluid measures the situation through hemato encephalic barrier through LC-MS.
Experimental result shows: conjugate I experimental group can detect Zorubicin in cerebrospinal fluid, and control group does not detect the existence of Zorubicin; Conjugate IV experimental group can detect taxol in cerebrospinal fluid, and control group does not detect the existence of taxol.
embodiment 3: conjugate is through the in-vitro evaluation of hemato encephalic barrier
In the present embodiment, the cell of employing is: Hela, SK-BR-3, NIH3T3 (purchased from Chinese Academy of Sciences's Shanghai life science institute biological chemistry and Institute of Cell Biology), by cell kind in 24 orifice plates, concentration is 2 × 10
5hole, overnight incubation, conjugate I and conjugate IV PBS dissolves, and makes its final concentration be 10umol/l with cell incubation.
In the present embodiment, use immunofluorescence dyeing, 1. use the formaldehyde of 4% 4 ° of lower fixed cells 10 minutes.2. use 3% cold Triton-X100 damping fluid 4 ° of permeation cells 20 minutes.3. the BSA Block buffer of 2% at room temperature closing cell 30 minutes.4. goat-anti people-FLAGmAb at room temperature incubated cell 1 hour.5. 2mg/mlStreptavidin-Alexa499 at room temperature incubated cell 1 hour.Below often walk and all rinse at least twice with PBS.6. 0.3%Triton-X100 damping fluid washes 20 minutes.Observe under the most rearmounted cell and microscope and confocal microscopy.
Experimental result shows: all have dyeing in cell, namely shows that conjugate I and conjugate IV is all by cytolemma.
embodiment 4: conjugate antitumor action is studied
In the present embodiment, the cell of employing is: FCY20201MCF-7, FCY202111590BS524 (T47D), TC-hxbz-286M453, utilizes mtt assay to evaluate the tumor killing effect of conjugate.
Collect logarithmic phase cell, adjustment concentration of cell suspension, every hole adds 100ul, and bed board makes cell to be measured adjust density to 1000-10000 hole.5%CO
2, hatch for 37 DEG C, be paved with (96 hole flat underside) at the bottom of hole, within 4 hours, add the medicine of 5 concentration gradients later to cell monolayer, in principle, every hole 100ul, if 5 multiple holes, continues to hatch 48 hours, observes under inverted microscope.Every hole adds 20ulMTT solution (5mg/ml, i.e. 0.5%MTT), continues to cultivate 4h.Carefully suck nutrient solution in hole, every hole adds 150ul dimethyl sulfoxide (DMSO), puts low-speed oscillation 5min on shaking table, crystallisate is fully dissolved.The light absorption value in each hole is measured at enzyme-linked immunosorbent assay instrument OD490nm place.Zeroing hole (substratum, MTT, dimethyl sulfoxide (DMSO)) is set simultaneously, control wells (the medicine dissolution medium of cell, same concentrations, nutrient solution, MTT, dimethyl sulfoxide (DMSO)).
The present embodiment calculation result adopts formula:
Proliferation inhibition rate=1-(experimental port A value-blank well A value)/(control wells A value-blank well A value), the A value wherein often organized is for deducting the result after withered hole.
Experimental result display: conjugate I and conjugate IV has good tumor killing effect, can calculate conjugate I according to proliferation inhibition rate formula and conjugate IV is respectively 99.98% at the tumour inhibiting rate of 100ug/ml, and 99.92%.99.92% is respectively, 98.15% at the tumour inhibiting rate of 10ug/ml.86.63% is respectively, 79.21% at the tumour inhibiting rate of 1ug/ml.20.88% is respectively, 6.4% at the tumour inhibiting rate of 1ug/ml.
Table 2: conjugate I absorbance
Table 3: conjugate IV absorbance
Claims (6)
1. a cell-penetrating peptide, is characterized in that, described cell-penetrating peptide is made up of 13 amino acid, and aminoacid sequence is:
RGGRVRRRWRGGR。
2. a conjugate, passes through formation of chemical bond by transmembrane polypeptide as claimed in claim 1 and antitumor drug.
3. conjugate according to claim 2, is characterized in that, described antitumor drug is selected from Zorubicin, taxol.
4. conjugate according to claim 2, is characterized in that described chemical bond is amido linkage.
5. prepare a method of wearing film peptide as claimed in claim 1, comprise and adopt the synthesis of Fmoc solid phase polypeptide synthesis.
6. cell-penetrating peptide as claimed in claim 1 is preparing the purposes in antitumor drug.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410281405.7A CN105315348B (en) | 2014-06-23 | 2014-06-23 | A kind of transmembrane polypeptide and its preparation method and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410281405.7A CN105315348B (en) | 2014-06-23 | 2014-06-23 | A kind of transmembrane polypeptide and its preparation method and application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105315348A true CN105315348A (en) | 2016-02-10 |
| CN105315348B CN105315348B (en) | 2018-10-16 |
Family
ID=55243738
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410281405.7A Active CN105315348B (en) | 2014-06-23 | 2014-06-23 | A kind of transmembrane polypeptide and its preparation method and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105315348B (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105418733A (en) * | 2015-06-23 | 2016-03-23 | 天津药物研究院有限公司 | Preparation and application of conjugate of cell penetrating polypeptide and antitumor drug |
| CN108059655A (en) * | 2017-12-25 | 2018-05-22 | 肽泽（武汉）生物科技有限公司 | A kind of cell-penetrating peptide and preparation method thereof, application |
| CN108707187A (en) * | 2018-06-12 | 2018-10-26 | 肽泽（武汉）生物科技有限公司 | A kind of cell-penetrating peptide and preparation method thereof, application |
| CN110680928A (en) * | 2019-12-06 | 2020-01-14 | 北京岳昊科技发展有限公司 | Conjugate for inhibiting melanin synthesis and application thereof in medicines and cosmetics |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1600372A (en) * | 2003-09-26 | 2005-03-30 | 王正荣 | Medication carrier of perforating membrane peptide of bell protein |
| WO2013098337A1 (en) * | 2011-12-27 | 2013-07-04 | Universite Pierre Et Marie Curie (Paris 6) | Cell-penetrating peptides |
| JP2013215104A (en) * | 2012-04-04 | 2013-10-24 | Okayama Univ | Polypeptide which selectively introduces adenovirus vector into cancer cell and adenovirus vector having the same |
-
2014
- 2014-06-23 CN CN201410281405.7A patent/CN105315348B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1600372A (en) * | 2003-09-26 | 2005-03-30 | 王正荣 | Medication carrier of perforating membrane peptide of bell protein |
| WO2013098337A1 (en) * | 2011-12-27 | 2013-07-04 | Universite Pierre Et Marie Curie (Paris 6) | Cell-penetrating peptides |
| JP2013215104A (en) * | 2012-04-04 | 2013-10-24 | Okayama Univ | Polypeptide which selectively introduces adenovirus vector into cancer cell and adenovirus vector having the same |
Non-Patent Citations (2)
| Title |
|---|
| JAKOB REGBERG ET AL.: "Applications of Cell-Penetrating Peptides for Tumor Targeting and Future Cancer Therapies", 《PHARMACEUTICALS》 * |
| 刘源等: "9R穿膜肽可增强P53抗肿瘤效果", 《生物工程学报》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105418733A (en) * | 2015-06-23 | 2016-03-23 | 天津药物研究院有限公司 | Preparation and application of conjugate of cell penetrating polypeptide and antitumor drug |
| CN108059655A (en) * | 2017-12-25 | 2018-05-22 | 肽泽（武汉）生物科技有限公司 | A kind of cell-penetrating peptide and preparation method thereof, application |
| CN108059655B (en) * | 2017-12-25 | 2021-01-22 | 肽泽（武汉）生物科技有限公司 | Cell-penetrating peptide and preparation method and application thereof |
| CN108707187A (en) * | 2018-06-12 | 2018-10-26 | 肽泽（武汉）生物科技有限公司 | A kind of cell-penetrating peptide and preparation method thereof, application |
| CN108707187B (en) * | 2018-06-12 | 2021-01-29 | 肽泽（武汉）生物科技有限公司 | Cell-penetrating peptide and preparation method and application thereof |
| CN110680928A (en) * | 2019-12-06 | 2020-01-14 | 北京岳昊科技发展有限公司 | Conjugate for inhibiting melanin synthesis and application thereof in medicines and cosmetics |
Also Published As
| Publication number | Publication date |
|---|---|
| CN105315348B (en) | 2018-10-16 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110114075B (en) | Disulfide-containing cell penetrating peptides and methods of making and using the same | |
| Brock | The uptake of arginine-rich cell-penetrating peptides: putting the puzzle together | |
| Han et al. | Bioconjugation of supramolecular metallacages to integrin ligands for targeted delivery of cisplatin | |
| KR20210055070A (en) | Il-2 receptor binding compounds | |
| US20150376237A1 (en) | Polypeptides for blood brain barrier transport | |
| Pilkington-Miksa et al. | Design, synthesis, and biological evaluation of novel cRGD–paclitaxel conjugates for integrin-assisted drug delivery | |
| CN105315348A (en) | Transmembrane polypeptide, preparation method and application thereof | |
| CN105418733A (en) | Preparation and application of conjugate of cell penetrating polypeptide and antitumor drug | |
| CN102105157A (en) | A conjugate of a polymer, an anti-angiogenesis agent and a targeting moiety, and uses thereof in the treatment of bone related angiogenesis conditions | |
| CN104774245A (en) | Preparation method and use of brain targeting peptide-antineoplastic drug conjugate | |
| CN103044522B (en) | Polypeptide having high affinity with receptor of integrin alpha v beta3 | |
| CN105198964A (en) | Tumor targeted polypeptide, and preparation method and application thereof | |
| US20200276323A1 (en) | Polypeptide conjugates for intracellular delivery of stapled peptides | |
| US10294260B2 (en) | Tetracyclic anthraquinone derivatives | |
| Burnett et al. | Synthesis, in vitro, and in vivo characterization of an integrin αvβ3-targeted molecular probe for optical imaging of tumor | |
| Lu et al. | Polymer chimera of stapled oncolytic peptide coupled with anti-PD-L1 peptide boosts immunotherapy of colorectal cancer | |
| CN105906692A (en) | cRGD-erlotinib conjugate and preparation method thereof | |
| CN105330725B (en) | A kind of pH response and polypeptide and its application for targeting human tumour vascular markers VEGFR2 | |
| CN103897043A (en) | Preparation and application of transmembrane polypeptide connected lapatinib | |
| CN104031119A (en) | Brain targeted peptide, and preparation method and application thereof | |
| CN113166188B (en) | Amphotericin B peptide derivatives | |
| Saraf et al. | Integrin targeting using RGD-based peptide amphiphiles | |
| Parrasia et al. | DA7R: a 7-letter Zip code to target PDAC | |
| Karakus et al. | Discovery of dual targeting PEGylated BG-P1600-TAT to norepinephrine transporter (NET) and thyrointegrin αvβ3 in the treatment of neuroblastoma | |
| CN104619697A (en) | Derivatives of 1-(substituted sulfonyl)-2-aminoimidazoline as antitumor and antiproliferative agents |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CP03 | Change of name, title or address | ||
| CP03 | Change of name, title or address |
Address after: 300301 No. 306, Huiren Road, Binhai Science Park, Binhai high tech Zone, Binhai New Area, Tianjin Patentee after: Tianjin Institute of Pharmaceutical Research Co.,Ltd. Address before: 300193 No. 308, Anshan West Road, Nankai District, Tianjin Patentee before: Tianjin Institute of Pharmaceutical Research |
|
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20211009 Address after: 300301 No. 308, Huiren Road, Binhai Science Park, Binhai high tech Zone, Binhai New Area, Tianjin Patentee after: Tianjin Tiancheng new drug evaluation Co.,Ltd. Address before: 300301 No. 306, Huiren Road, Binhai Science Park, Binhai high tech Zone, Binhai New Area, Tianjin Patentee before: Tianjin Institute of Pharmaceutical Research Co.,Ltd. |