CN106589062A - Grb2 protein targeting anti-tumor covalent polypeptide preparation and preparation method and application thereof - Google Patents
Grb2 protein targeting anti-tumor covalent polypeptide preparation and preparation method and application thereof Download PDFInfo
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- CN106589062A CN106589062A CN201611128097.XA CN201611128097A CN106589062A CN 106589062 A CN106589062 A CN 106589062A CN 201611128097 A CN201611128097 A CN 201611128097A CN 106589062 A CN106589062 A CN 106589062A
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Life Sciences & Earth Sciences (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention relates to a Grb2 protein targeting anti-tumor covalent polypeptide preparation and a preparation method and application thereof; the amino acid sequence of the covalent polypeptide preparation is VPPPVPPRRX, wherein X is (2S)-2-amino-3-[(2-chloracetyl)amino]propionic acid. The preparation method is Fmoc solid polypeptide synthetic method, and X is made from Fmoc-Dap(Mtt)-OH and chloroacetic acid. The covalent polypeptide inhibitor of the invention has high affinity to Grb2 protein and thereby is capable of avoiding 'off-target covalent reaction; therefore, the covalent polypeptide inhibitor has good treatment effect, and the problem that the prior art covalent polypeptide drugs have toxic and side effects is solved. A new idea to research and develop covalent inhibitor drugs is provided, and the treatment of diseases, such as breast cancer, is facilitated.
Description
Technical field
The present invention relates to molecular biology and pharmaceutical technology field, specifically, are related to a kind of the anti-of targeting Grb2 albumen
Covalent peptide inhibitor of tumor and its preparation method and application.
Background technology
Covalency inhibitor is uniquely can silence or the permanent class medicine for closing pathogenic protein completely in existing Pharmacological inhibitors
Thing, its mechanism of action be covalency inhibitor can with target protein specific some or several amino acid residues occur it is covalently anti-
Should, so as to suppress the related activity of target protein, the purpose for the treatment of disease is reached with this.Compared to conventional noncovalent drug, altogether
Valency drug dose is few and effect is lasting.U.S. FDA and China CFDA have passed through the examination ＆ approval of some such medicines and have applied in recent years
In clinic.Due to the unique treatment advantage of covalency inhibitor, it is also used in oncotherapy research, and it is aobvious to have obtained some
The effect of work.If Neubig and its colleague are using one compound of a pearl (one-bead one-compound, OBOC) storehouse screening side
Method has been obtained one and can have been reached by covalently suppressing RGS4 with the covalently bound polypeptide of regulation G-protein signal protein 4 (RGS4)
The purpose of modulate tumor associated protein G-protein, and then suppress g protein coupled receptor coherent signal.Taunton and its colleague's exploitation
One kind with the covalently bound covalency inhibitors of ribosomal protein kinases RSK2 in breast cancer cell MDA-MB-231, and can be obtained
Good tumor killing effect.Although covalent drug shows powerful effect in terms of protein biological activity is suppressed, some are covalent
Inhibitor can be reacted with non-specific albumen in cell, and this " missing the target " covalent reaction can bring very strong poison secondary to body
Effect, seriously hinders the development of covalency inhibitor.
Growth factor receptor binding protein precursor 2 (Growth factor receptor bound protein 2, Grb2) is
One class adaptin, its SH2 (Src homology 2) domain and the SH3 (Src positioned at two ends by a centre
3) domain is constituted homology.Research discovery Grb2 in high expression status in breast carcinoma, carried down with lymph by its expression
Move and prognosis is related.In breast cancer cell, Grb2 is interacted with Protein S os (son of sevenless) and by regulation and control
Receptor tyrosine kinase (receptor tyrosine kinase, RTK) signal path come promote tumor cell invasion and attack and turn
Move.
The covalency inhibitor class medicine of targeting Grb2 albumen equally exists " missing the target " problem in treatment breast carcinoma, causes medicine
Thing toxic and side effects are larger, and therapeutic effect is not good enough.As can be seen here, it is necessary to design is a kind of specifically occur with Grb2 covalent reaction,
It is difficult the medicine for missing the target.
The content of the invention
The purpose of the present invention is for deficiency of the prior art, there is provided a kind of antitumor of targeting Grb2 albumen is covalently more
Inhibitor peptides.
Another purpose of the present invention is to provide the preparation method of described covalent peptide inhibitor.
Another purpose of the present invention is to provide the purposes of described covalent peptide inhibitor.
To realize above-mentioned first purpose, the present invention is adopted the technical scheme that：
A kind of covalent peptide inhibitor of antineoplastic of targeting Grb2 albumen, the aminoacid of described covalent peptide inhibitor
Sequence is：VPPPVPPRRX, wherein X are (2S) -2- amino -3- [(2- chloracetyls) amino] propanoic acid.
To realize above-mentioned second purpose, the present invention is adopted the technical scheme that：
The preparation method of described covalent peptide inhibitor, described preparation method are Fmoc solid phase polypeptide synthesis.
The raw material of X is Fmoc-Dap (Mtt)-OH and monoxone.
To realize above-mentioned 3rd purpose, the present invention is adopted the technical scheme that：
Application of the described covalent peptide inhibitor in the medicine for preparing the protein mediated diseases for the treatment of Grb2.
Described Grb2 protein mediated disease is tumor.
Described tumor is breast carcinoma.
The invention has the advantages that：
Present inventor has found the N-terminal SH3 domain (Grb2 in Grb2 in research processN-SH3, PDB:1GBQ)
In have a cysteine residues (Cys32) and its have the mercapto groups of reactivity be exposed to outside, very close to it
Interaction protein Sos.The polypeptide fragment VPPPVPPRR in Sos sourcesRThere are higher affinity, and this fragment C-terminal with Grb2
Arg (bottom indicates horizontal line) and Grb2N-SH3Cys32 closely, then with an alpha-non-natural amino acid X (carry nucleophilic group,
Wherein X is (2S) -2- amino -3- [(2- chloracetyls) amino] propanoic acid) replace Arg, X can be with the N-terminal SH3 domains of Grb2 albumen
In Cys32 there is nucleophilic substitution, such that it is able to this peptide inhibitor is covalently bound on Grb2 albumen, can be with this
The signal path in regulation and control Grb2 downstreams is reached, and then suppresses the purpose of growth of tumour cell.This new polypeptide VPPPVPPRRX
The covalency inhibitor of (being named as RP) as Grb2, is unique a kind of covalent polypeptide for breast cancer cell Grb2 albumen so far
Inhibitor.Because RP and Grb2 has high affinity, Gu Qihui avoids " missing the target " covalent reaction, is reaching suppression breast cancer cell
Other toxic and side effects will not be also produced while growth.The present invention provides a new approaches for covalency inhibitor medicament research and development,
Will be helpful to the treatment of breast carcinoma.
Description of the drawings
Fig. 1. the chemical structural formula of covalent peptide inhibitor RP.
The Mass Spectrometric Identification result of Fig. 2 .RP sequences VPPPVPPRRX.
Fig. 3 .RP and Grb2N-SH3The covalently bound mass spectral results of albumen.
Impact testing results of Fig. 4 .RP to breast cancer cell growth activity.
The impact testing result that Fig. 5 .RP are migrated to breast cancer cell.
Impact testing results of Fig. 6 .RP to Apoptosis of Breast Cancer.
Specific embodiment
Below in conjunction with the accompanying drawings the specific embodiment that the present invention is provided is elaborated.
Embodiment 1
First, the synthesis of covalent peptide inhibitor RP and sign
1 method
The synthesis of 1.1 covalent peptide inhibitor RP
1) using Fmoc chemical solid phase Peptide systhesis, using Rink Amide-ChemMatrix resins, with 5 times of excess
The aminoacid and condensation reagent (HBTU/HOBt) synthesis polypeptide of Fmoc protections, the wherein precursor of X Fmoc-Dap (Mtt)-OH.
2) protected with acetic anhydride after first aminoacid Fmoc deprotection of polypeptide N-terminal.
3) by the Mtt of X precursors 1%TFA selectivity deprotections, monoxone is added, side chain is modified into into acyl chlorides.
4) polypeptide is cut off from resin with 95%TFA, is precipitated with ice ether, the preliminary polypeptide HPLC for obtaining separates pure
Change.
The sign of 1.2 covalent peptide inhibitor RP
The polypeptide that Jing HPLC are isolated and purified detects confirmation with MALDI-TOF MS.
2 results
Covalently the sequence of peptide inhibitor RP is VPPPVPPRRX, and wherein X is (2S) -2- amino -3- [(2- chloracetyls) ammonia
Base] propanoic acid.Covalently the chemical structural formula of peptide inhibitor RP is shown in Fig. 1.The Mass Spectrometric Identification result of RP sequences VPPPVPPRRX is shown in figure
2。
2nd, covalent peptide inhibitor RP and Protein G rb2N-SH3External covalent bond detection
1 method
1.1 build pGEX-6P-3-Grb2N-SH3Prokaryotic expression plasmid
The total serum IgE of breast cancer cell SK-BR-3 is extracted with Trizol, afterwards with PrimeScript RT reagent Kit
With gDNA Eraser (Takara) test kit reverse transcriptions go out first chain cDNA, synthesize Grb2 with PCRN-SH3Fragment (SEQ
ID NO.1) and verified with agarose gel electrophoresiies, sequencing afterwards determines.Jing after sequencing determines, fragment T4 ligases
(Takara) prokaryotic expression plasmid pGEX-6P-3 (between NdeI and XhoI) and then digestion verification are connected.
1.2 E.Coli prokaryotic expressions purifying protein Grb2N-SH3
The correct pGEX-6P-3-Grb2 of empirical testsN-SH3Plasmid is converted in BL-21 competent cells, is just choosing clone's checking
After really, it is added in the 5mL LB culture medium of sterilizing 37 DEG C and shakes overnight.4mL is taken afterwards be added in 400mL LB 37 DEG C shake OD600About
04-0.6, plus IPTG inductions, then 16 DEG C are shaken 18 hours.Centrifuge removes bacterium solution supernatant, thalline ultrasonication afterwards.Utilize
GST label purifying proteins, and GST labels are cut off with PreScission enzymes (GE Healthcare Life Sciences), obtain
To Protein G rb2N-SH3And verified with SDS-PAGE.
1.3 detection RP and Grb2N-SH3Albumen covalent bond
RP and Grb2N-SH3Albumen presses 5:1 amount hybrid reaction 30 minutes.After negating the mixture heat denatured desalination answered,
With MALDI-TOF MS precise verification RP and Grb2N-SH3Albumen whether covalent bond.
2 results
RP and Grb2N-SH3The covalently bound Mass Spectrometric Identification result of albumen is shown in Fig. 3, as a result shows RP and Grb2N-SH3Albumen is sent out
Covalent bond is given birth to.
3rd, RP antitumous effects are detected in breast cancer cell line
1 method
Impact detections of the 1.1RP to breast cancer cell growth activity
Breast cancer cell SK-BR-3 is laid on 96 orifice plates, per 3000, hole cell.Plus with cell-penetrating peptide (YGRKKRRQRRR,
SEQ ID NO.2) RP and with cell-penetrating peptide it is non-covalent control polypeptide (VPPPVPPRRR, SEQ ID NO.3), concentration be 40 μ
M.24 hours, 48 hours and 72 hours three time points are cultivated afterwards, suction out culture fluid, plus MTT solution, cell culture incubator culture
Detected with microplate reader after 2 hours.
The impact detection that 1.2 RP are migrated to breast cancer cell
Breast cancer cell SK-BR-3 is laid on 24 orifice plates, per hole 1 × 105Individual cell.Plus all carry above-mentioned cell-penetrating peptide RP and
Non-covalent control polypeptide, concentration are 40 μM, after 8 hours.Under cell is digested with pancreatin, and hole on Transwell is added on, upper hole is used
Serum-free medium, lower opening suction out culture fluid with 10% serum free culture system liquid after cultivating 18 hours.Washed after twice with PBS, with knot
Crystalviolet staining cell 30 minutes, is cleaned with PBS afterwards, is counted under the microscope and is colored number of cells.
Impact detections of 1.3 RP to Apoptosis of Breast Cancer
Breast cancer cell SK-BR-3 is laid on 24 orifice plates, per hole 1 × 105Individual cell.Plus all carry above-mentioned cell-penetrating peptide RP and
Non-covalent control polypeptide, concentration are 40 μM, cultivate 24 hours, 48 hours and 72 hours three time points afterwards, suction out culture fluid,
After with the lower cell of pancreatin digestion, dyeed with Annexin V/FITC apoptosis test kits (Sigma-Aldrich), it is thin with streaming afterwards
Born of the same parents' instrument is detected.
2 results
Impacts of 2.1 RP to breast cancer cell growth activity
See Fig. 4, as a result show that RP can significantly inhibit the growth activity of breast cancer cell.
The impact that 2.2 RP are migrated to breast cancer cell
See Fig. 5, as a result show that RP can significantly inhibit the migration of breast cancer cell.
Impacts of 2.3 RP to Apoptosis of Breast Cancer
See Fig. 6, as a result show that RP can remarkably promote the apoptosis of breast cancer cell.
The above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
Member, on the premise of without departing from the inventive method, can also make some improvement and supplement, and these improve and supplement also should be regarded as
Protection scope of the present invention.
SEQUENCE LISTING
<110>The first maternity and infant health institute, Shanghai City
<120>Covalent peptide inhibitor of antitumor of targeting Grb2 albumen and its preparation method and application
<130> /
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 201
<212> DNA
<213>Artificial sequence
<400> 1
catatgatgg aagccatcgc caaatatgac ttcaaagcta ctgctgacga tgagctgagc 60
ttcaaaaggg gggacatcct taaggttttg aatgaagaat gtgaccagaa ctggtataag 120
gcagaactca atgggaaaga tggcttcatc cccaagaatt acatagaaat gaaaccacat 180
actcgagcgg ccgcatcgtg a 201
<210> 2
<211> 11
<212> PRT
<213>Artificial sequence
<400> 2
Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg
1 5 10
<210> 3
<211> 10
<212> PRT
<213>Artificial sequence
<400> 3
Val Pro Pro Pro Val Pro Pro Arg Arg Arg
1 5 10
Claims (6)
1. the covalent peptide inhibitor of antineoplastic of a kind of targeting Grb2 albumen, it is characterised in that described covalent polypeptide suppresses
The aminoacid sequence of agent is：VPPPVPPRRX, wherein X are (2S) -2- amino -3- [(2- chloracetyls) amino] propanoic acid.
2. the preparation method of the covalent peptide inhibitor described in claim 1, it is characterised in that described preparation method is Fmoc
Solid phase polypeptide synthesis.
3. preparation method according to claim 2, it is characterised in that the raw material of X is Fmoc-Dap (Mtt)-OH and chloroethene
Acid.
4. the covalent peptide inhibitor described in claim 1 in the medicine for preparing the treatment protein mediated diseases of Grb2 should
With.
5. application according to claim 4, it is characterised in that described Grb2 protein mediated disease is tumor.
6. application according to claim 5, it is characterised in that described tumor is breast carcinoma.
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Cited By (2)
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| CN108640974A (en) * | 2018-05-19 | 2018-10-12 | 上海市第妇婴保健院 | A kind of polypeptide and its dimer of targeting Syntenin albumen PDZ structural domains |
| CN110669146A (en) * | 2019-10-24 | 2020-01-10 | 常州大学 | Active peptide capable of specifically blocking binding site of adaptor protein Nck and used for preventing intestinal tract EPEC infection |
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| EP1281963A2 (en) * | 2001-07-30 | 2003-02-05 | Warner-Lambert Company | Method for the screening of compounds that inhibit the interaction between a proline-rich peptide and a SH3 domain comprising peptide |
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| EP1281963A2 (en) * | 2001-07-30 | 2003-02-05 | Warner-Lambert Company | Method for the screening of compounds that inhibit the interaction between a proline-rich peptide and a SH3 domain comprising peptide |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108640974A (en) * | 2018-05-19 | 2018-10-12 | 上海市第妇婴保健院 | A kind of polypeptide and its dimer of targeting Syntenin albumen PDZ structural domains |
| CN108640974B (en) * | 2018-05-19 | 2021-07-06 | 上海市第一妇婴保健院 | Polypeptide of targeting Syntenin protein PDZ structural domain and dimer thereof |
| CN110669146A (en) * | 2019-10-24 | 2020-01-10 | 常州大学 | Active peptide capable of specifically blocking binding site of adaptor protein Nck and used for preventing intestinal tract EPEC infection |
| CN110669146B (en) * | 2019-10-24 | 2023-05-23 | 常州大学 | Active peptide capable of specifically blocking binding site of adapter protein Nck for preventing intestinal EPEC infection |
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| CN106589062B (en) | 2020-03-24 |
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