CN105315348B - A kind of transmembrane polypeptide and its preparation method and application - Google Patents
A kind of transmembrane polypeptide and its preparation method and application Download PDFInfo
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- CN105315348B CN105315348B CN201410281405.7A CN201410281405A CN105315348B CN 105315348 B CN105315348 B CN 105315348B CN 201410281405 A CN201410281405 A CN 201410281405A CN 105315348 B CN105315348 B CN 105315348B
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Abstract
The invention belongs to pharmaceutical technology fields, provide a kind of cell-penetrating peptide, are made of 13 amino acid, sequence RGGRVRRRWRGGR.The present invention obtains the polypeptide by the method for synthesis in solid state, and is coupled by chemically synthesized method and antitumor drug, and conjugate provided by the present invention has the function of passing through blood-brain barrier and tumor-inhibiting action.
Description
Technical field
The invention belongs to pharmaceutical technology fields, are related to the relevant drug field of tumour, specifically, the present invention relates to one kind
Transmembrane polypeptide and the polypeptide are coupled the conjugate to be formed with antineoplastic.
Background technology
The incidence of brain tumor and metastatic encephaloma rises year by year.Aspect is being treated to it, blood-brain barrier is considered as preventionization
Treat the main reason for drug enters brain tissue and then affects the treatment.Blood-brain barrier be body participate in inherent immunity internal barrier it
One, the brain capillary wall by pia mater, choroid plexus between blood circulation and brain parenchym and the cutose institute group that is wrapped in outside wall
At can stop causal organism and other macromolecular substances enter brain tissue and the ventricles of the brain by blood circulation.Substantially 100% macromolecular
Drug, including polypeptide, recombinant protein, monoclonal antibody, the drug based on RNA perturbation techniques, gene therapy related drugs etc. are all
Blood-brain barrier can not be passed through, and the small-molecule drug more than 98% can not also pass through blood-brain barrier.Currently, radiation treatment and change
Patient vitals 4 months can only be extended by learning therapy, therefore this invention address that research makes existing drug preferably penetrate blood-brain barrier.
Polypeptide has higher availability, due to itself in vivo since it is with polar terminals and non-polar end
It is endogenous material so small toxicity, but polypeptide is limited directly as drug by larger, such as its stability is poor, it cannot
It is oral.In recent years, polypeptide has been increasingly becoming one of the hot spot of new drug development as the application of drug miscellaneous function.Cell-penetrating peptide
It is 20th century mid-term peptide molecule of a kind of tool specific function that starts to recognize, English science title is generally written as cell2-
penetrating-peptides(CPPs).They are usually made of 5~30 amino acid, typically generally by 8~16 ammonia
Base acid is constituted, and has the function of membranes penetration, can also be carried other molecules even supermolecule particle and be entered in cell.Exactly because
This special function, they are counted as the effective intracellular transport tool of bioactive molecule, are with a wide range of applications.
Polypeptide and drug were many by the example of covalent manner formation composition in recent years, such as：Polypeptide
Penetratin and adriamycin are covalently coupled to treatment breast cancer；Peptide T AT and drug P53 is covalently coupled to treatment cancer eye
Transfer；Polypeptide YTA4 covalently couples treatment breast cancer with methotrexate (MTX).
Invention content
It is the detailed description of the present invention below：
(1) cell-penetrating peptide of the present invention
Cell-penetrating peptide of the present invention is made of 13 amino acid, and is hydrophily and hydrophobicity alternate combinations
, amino acid sequence is：
RGGRVRRRWRGGR
The polypeptide itself does not have physiological activity, but has the function of penetrating blood-brain barrier, and it is more that the invention further relates to this
The preparation method and applications of peptide.
(2) preparation method of cell-penetrating peptide of the present invention
Polypeptide needed for the present invention is prepared using the method for solid phase, synthesis in solid state be using solid support resin as carrier,
The process of amino acid is gradually added from C-terminal, that is, c-terminus to N-terminal, that is, aminoterminal.
Preferred resin in the present invention：Fmoc-wang resins, 2-cl-trt resins, 0.24~0.4. of resin degree of substitution
Protected amino acid used in the present invention is：Fmoc-Arg (pbf)-OH, Fmoc-Gly-OH, Fmoc-Val-OH,
Fmoc-Trp-OH, 3~5 times of excess of protected amino acid in reaction process.
Deprotecting regent used in the present invention is：Piperidines/n,N-Dimethylformamide, ratio 10:90~30:70.
Coupling reagent used in the present invention is：1. benzotriazole-N, N, N', N'- tetramethylurea hexafluorophosphate/1-
Hydroxybenzotriazole (HOBT), 2. 2- (7- azos benzotriazole)-N, N, N', N'- tetramethylureas hexafluorophosphoric acid ester/1- hydroxyls
Benzotriazole (HOBT), 3. N, N'- diisopropylcarbodiimide/I-hydroxybenzotriazole (HOBT).
In the present invention, if 1. or 2. the plants coupling reagent using the, then acid binding agent should be added during in reaction：
DIEA/NMP。
Cutting reagent used in the present invention is：TFA/ thioanisole/water/phenol/1,2- dithioglycol, ratio are：
82.5:5:5:5；2.5.
MS-IT-TOF used in the present invention determines molecular weight, and thick peptide is purified using HPLC
(3) connection method of polypeptide of the present invention and antitumor drug adriamycin
1) synthesis of compound II
Addition 1.0g (1.72mmol) adriamycin in 50m reaction bulbs, 0.52g (5.20mmol) succinic anhydride, 30mlTHF,
0.66g (5.12mmol) n,N-diisopropylethylamine (DIEA), 27 DEG C of stirring 6h is added dropwise, solvent evaporated obtains wax, water is added to stir
It mixes, there is yellow solid precipitation, filter, washing is twice, dry, obtains yellow solid 1.15g.
2) synthesis of compound I
0.22g (0.32mmol) compounds II, 0.10g (0.31mmol) O- benzotriazole-N are added in 50m reaction bulbs,
It is different that 0.08g (0.62mmol) N, N- bis- is added dropwise in N, N', N'- tetramethylurea tetrafluoro boric acid ester (TBTU), 5mlDMF, nitrogen protection
Propylethylamine (DIEA) after 27 DEG C are stirred 1h, is added 0.10g polypeptides, after 27 DEG C are stirred 3h, terminates reaction.Use MS-IT-TOF
It determines molecular weight, uses HPLC purification of crude product.
(4) connection method of polypeptide of the present invention and antitumor drug paclitaxel
1) synthesis of compound III
Addition 1.50g (1.72mmol) taxol in 50m reaction bulbs, 0.52g (5.20mmol) succinic anhydride, 30mlTHF,
0.66g (5.12mmol) n,N-diisopropylethylamine (DIEA), 27 DEG C of stirring 6h is added dropwise, solvent evaporated obtains wax, water is added to stir
It mixes, there is yellow solid precipitation, filter, washing is twice, dry, obtains yellow solid 1.15g.
2) synthesis of compound IV
0.22g (0.32mmol) compound III, 0.10g (0.31mmol) O- benzotriazole-is added in 50m reaction bulbs
0.08g (0.62mmol) N, N- bis- is added dropwise in N, N, N', N'- tetramethylurea tetrafluoro boric acid ester (TBTU), 5mlDMF, nitrogen protection
Wopropyl ethyl amine (DIEA) after 27 DEG C are stirred 1h, is added 0.10g polypeptides, after 27 DEG C are stirred 3h, terminates reaction.Use MS-IT-
TOF determines molecular weight, uses HPLC purification of crude product.
Description of the drawings
Fig. 1 is the HPLC test result figures of synthesis polypeptide.
Specific implementation mode
The present invention is further described in detail With reference to embodiment, the embodiment provided is only for explaining
The bright present invention, the range being not intended to be limiting of the invention.
Below in an example, the various processes and method not being described in detail are conventional methods as known in the art.
Embodiment 1:The synthesis in solid state of polypeptide
Using the solid-phase peptide synthesis (amino acid used is purchased from Shanghai gill company) of Fmoc strategies, CEM is used
The Liberitity type instruments of company's production carry out the synthesis of the transmembrane polypeptide of the present invention.Operating method according to manufacturer instrument
Specification carries out.
Synthesize agents useful for same selection：
(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, 5 times of excess of protected amino acid used in reaction.
(3) deprotecting regent used in the present invention is：Piperidines/n,N-Dimethylformamide, ratio 20:80.
(4) coupling reagent used in the present invention is：N, N'- diisopropylcarbodiimide (DIC)/I-hydroxybenzotriazole
(HOBT)。
(5) cutting reagent used in the present invention is：TFA/ thioanisole/water/phenol/1,2- dithioglycol, ratio
For：82.5:5:5:5；2.5.
Polypeptide obtained is purified, testing conditions using HPLC C18 semi-preparative columns (being purchased from Phenomenex companies)
For：Detection wavelength：214nm, mobile phase A：Acetonitrile (contains 0.1% trifluoroacetic acid), Mobile phase B：Water (contains 0.1% trifluoroacetic acid),
Elution requirement：By 35%A~50%A in 30 minutes, determines gained sterling molecular weight using MS-IT-TOF, sample is determined with HPLC
Product purity, purity are more than 95% and obtain polypeptide freeze-dried powder through desalination and freeze-drying.
HPLC test results are shown in Fig. 1, table 1.
Table 1HPLC test results
Embodiment 2：Conjugate penetrates the interior evaluating of blood-brain barrier
By above-mentioned conjugate I and control adriamycin, conjugate IV and control taxol are configured to 10mg/ with physiological saline
Rat (being purchased from Chinese Academy of Sciences's Shanghai Experimental Animal Center, SD rats) is anesthetized with ether, through vena femoralis injection (200 μ by ml solution
L/, 6/group), cerebrospinal fluid is taken within 30 minutes after injection, and disconnected neck puts to death rat.Cerebrospinal fluid measures saturating through LC-MS
The case where crossing blood-brain barrier.
The experimental results showed that：Conjugate I experimental groups can detect adriamycin in cerebrospinal fluid, and there is no detect for control group
To the presence of adriamycin；Conjugate IV experimental groups can detect taxol in cerebrospinal fluid, and control group does not detect purple
The presence of China fir alcohol.
Embodiment 3：Conjugate penetrates the in-vitro evaluation of blood-brain barrier
In the present embodiment, the cell that uses for：Hela, SK-BR-3, NIH3T3 (are ground purchased from Chinese Academy of Sciences's Shanghai life science
Study carefully institute's biochemistry and Institute of Cell Biology), by cell kind in 24 orifice plates, a concentration of 2 × 105Hole, overnight incubation are even
Connection object I and conjugate IV is dissolved with PBS, makes its final concentration of 10umol/l with cell incubation.
In the present embodiment, using immunofluorescence dyeing, cell 10 minutes are 1. fixed under 4 ° with 4% formaldehyde.2. with 3%
Cold Triton-X100 buffer solutions were in 4 ° of permeation cells 20 minutes.3. 2% BSA Block buffers closing cell 30 at room temperature
Minute.4. goat-anti people-FLAG mAb are incubated at room temperature cell 1 hour.5. 2mg/ml Streptavidin-Alexa499 exist
Incubated cell 1 hour at room temperature.It often walks and is rinsed at least twice with PBS above.6. 0.3%Triton-X100 buffer solutions wash 20
Minute.Most postposition cell is observed under microscope and confocal microscopy.
The experimental results showed that：There is dyeing into the cell, that is, shows that conjugate I and conjugate IV can pass through cell membrane.
Embodiment 4：Conjugate antitumor action is studied
In the present embodiment, the cell that uses for：FCY20201 MCF-7, FCY20211 1590BS524 (T47D), TC-
Hxbz-286M453 evaluates the tumor killing effect of conjugate using mtt assay.
Logarithmic phase cell is collected, concentration of cell suspension is adjusted, 100ul is added per hole, bed board makes cell tune density to be measured extremely
The holes 1000-10000.5%CO2, 37 DEG C of incubations, until cell monolayer is paved with bottom hole (96 hole flat underside), addition 5 after 4 hours
The drug of concentration gradient per hole 100ul, if 5 multiple holes, continues to be incubated 48 hours, be observed under inverted microscope in principle.Often
20ulMTT solution (5mg/ml, i.e. 0.5%MTT) is added in hole, continues to cultivate 4h.Culture solution in hole carefully is sucked, is added per hole
150ul dimethyl sulfoxide (DMSO)s set low-speed oscillation 5min on shaking table, crystal are made fully to dissolve.In enzyme-linked immunosorbent assay instrument OD490nm
Place measures the light absorption value in each hole.Zeroing hole (culture medium, MTT, dimethyl sulfoxide (DMSO)), control wells (cell, same concentrations are set simultaneously
Drug dissolving medium, culture solution, MTT, dimethyl sulfoxide (DMSO)).
The present embodiment result of calculation uses formula：
Proliferation inhibition rate=1- (experimental port A values-blank well A values)/(control wells A values-blank well A values), wherein every group of A
Value is the result subtracted after withered hole.
Experimental result is shown：Conjugate I and conjugate IV has preferable tumor killing effect, can according to proliferation inhibition rate formula
It is respectively 99.98%, 99.92% to calculate conjugate I and conjugate IV in the tumour inhibiting rate of 100ug/ml.10ug/ml's
Tumour inhibiting rate is respectively 99.92%, 98.15%.It is respectively 86.63%, 79.21% in the tumour inhibiting rate of 1ug/ml.1ug/ml's
Tumour inhibiting rate is respectively 20.88%, 6.4%.
Table 2：Conjugate I absorbance values
Table 3：Conjugate IV absorbance values
Claims (5)
1. a kind of cell-penetrating peptide, which is characterized in that the cell-penetrating peptide is made of 13 amino acid, and amino acid sequence is：
RGGRVRRRWRGGR。
2. a kind of conjugate passes through formation of chemical bond by transmembrane polypeptide as described in claim 1 and antitumor drug.
3. conjugate according to claim 2, which is characterized in that the antitumor drug is selected from adriamycin, taxol.
4. conjugate according to claim 2, it is characterised in that the chemical bond is amido bond.
5. a kind of method preparing cell-penetrating peptide as described in claim 1, including synthesized using Fmoc solid phase polypeptide synthesis.
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| 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 |
| 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 |
| 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 |
| CN110680928B (en) * | 2019-12-06 | 2020-04-28 | 深圳前海港影生物科技有限公司 | Conjugate for inhibiting melanin synthesis and application thereof in medicines and cosmetics |
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| 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 |
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|---|---|---|---|---|
| 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 |
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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 |
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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. |