CN117756905A - Staple peptide and pharmaceutical application thereof - Google Patents
Staple peptide and pharmaceutical application thereof Download PDFInfo
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- CN117756905A CN117756905A CN202311782032.7A CN202311782032A CN117756905A CN 117756905 A CN117756905 A CN 117756905A CN 202311782032 A CN202311782032 A CN 202311782032A CN 117756905 A CN117756905 A CN 117756905A
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- Prior art keywords
- amino acid
- peptide
- flgailkighalaktvlpmvtnafkpkq
- replacing
- cyclization
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- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 112
- 102000004196 processed proteins & peptides Human genes 0.000 claims abstract description 46
- 125000000539 amino acid group Chemical group 0.000 claims abstract description 22
- 229920001184 polypeptide Polymers 0.000 claims abstract description 20
- 241000894006 Bacteria Species 0.000 claims abstract description 18
- 239000003814 drug Substances 0.000 claims abstract description 17
- 108090000631 Trypsin Proteins 0.000 claims abstract description 14
- 102000004142 Trypsin Human genes 0.000 claims abstract description 14
- 239000012588 trypsin Substances 0.000 claims abstract description 14
- 108090000317 Chymotrypsin Proteins 0.000 claims abstract description 13
- 229960002376 chymotrypsin Drugs 0.000 claims abstract description 13
- 241000589517 Pseudomonas aeruginosa Species 0.000 claims abstract description 7
- 241000588626 Acinetobacter baumannii Species 0.000 claims abstract description 5
- 241000191967 Staphylococcus aureus Species 0.000 claims abstract description 5
- 238000007363 ring formation reaction Methods 0.000 claims description 23
- 238000002360 preparation method Methods 0.000 claims description 7
- 230000000844 anti-bacterial effect Effects 0.000 claims description 6
- 238000004458 analytical method Methods 0.000 claims description 4
- 241000588724 Escherichia coli Species 0.000 claims description 3
- 125000003275 alpha amino acid group Chemical group 0.000 abstract description 27
- 150000001413 amino acids Chemical class 0.000 abstract description 8
- 230000002401 inhibitory effect Effects 0.000 abstract description 4
- 201000010099 disease Diseases 0.000 abstract description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 abstract description 3
- 238000012986 modification Methods 0.000 abstract description 3
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- 241000588722 Escherichia Species 0.000 abstract 1
- 229940121657 clinical drug Drugs 0.000 abstract 1
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 29
- 238000004128 high performance liquid chromatography Methods 0.000 description 25
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 20
- 238000000746 purification Methods 0.000 description 16
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- 229940079593 drug Drugs 0.000 description 13
- 238000001819 mass spectrum Methods 0.000 description 12
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- 102000035195 Peptidases Human genes 0.000 description 10
- 108091005804 Peptidases Proteins 0.000 description 10
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- 238000002474 experimental method Methods 0.000 description 10
- 239000012071 phase Substances 0.000 description 9
- 239000007788 liquid Substances 0.000 description 8
- DTQVDTLACAAQTR-UHFFFAOYSA-N trifluoroacetic acid Substances OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 7
- 125000003088 (fluoren-9-ylmethoxy)carbonyl group Chemical group 0.000 description 6
- 230000001580 bacterial effect Effects 0.000 description 6
- 230000000694 effects Effects 0.000 description 6
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 5
- 238000000034 method Methods 0.000 description 5
- 238000000926 separation method Methods 0.000 description 5
- 230000006641 stabilisation Effects 0.000 description 5
- 238000011105 stabilization Methods 0.000 description 5
- WSLDOOZREJYCGB-UHFFFAOYSA-N 1,2-Dichloroethane Chemical compound ClCCCl WSLDOOZREJYCGB-UHFFFAOYSA-N 0.000 description 4
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine (DIPEA) Chemical compound CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 description 4
- 230000015556 catabolic process Effects 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- 238000006731 degradation reaction Methods 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- WFDIJRYMOXRFFG-UHFFFAOYSA-N Acetic anhydride Chemical compound CC(=O)OC(C)=O WFDIJRYMOXRFFG-UHFFFAOYSA-N 0.000 description 3
- 239000003242 anti bacterial agent Substances 0.000 description 3
- 229940088710 antibiotic agent Drugs 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 230000002797 proteolythic effect Effects 0.000 description 3
- 238000005086 pumping Methods 0.000 description 3
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- 239000006228 supernatant Substances 0.000 description 3
- 208000035143 Bacterial infection Diseases 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 2
- NQRYJNQNLNOLGT-UHFFFAOYSA-N Piperidine Chemical compound C1CCNCC1 NQRYJNQNLNOLGT-UHFFFAOYSA-N 0.000 description 2
- 238000007792 addition Methods 0.000 description 2
- 229940124350 antibacterial drug Drugs 0.000 description 2
- 208000022362 bacterial infectious disease Diseases 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
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- 238000011161 development Methods 0.000 description 2
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- 238000000338 in vitro Methods 0.000 description 2
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- CMWYAOXYQATXSI-UHFFFAOYSA-N n,n-dimethylformamide;piperidine Chemical compound CN(C)C=O.C1CCNCC1 CMWYAOXYQATXSI-UHFFFAOYSA-N 0.000 description 2
- 239000003910 polypeptide antibiotic agent Substances 0.000 description 2
- 125000006239 protecting group Chemical group 0.000 description 2
- 238000004007 reversed phase HPLC Methods 0.000 description 2
- 238000010532 solid phase synthesis reaction Methods 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- BDNKZNFMNDZQMI-UHFFFAOYSA-N 1,3-diisopropylcarbodiimide Chemical compound CC(C)N=C=NC(C)C BDNKZNFMNDZQMI-UHFFFAOYSA-N 0.000 description 1
- JWZFECWHKQLRGK-UHFFFAOYSA-N 2-amino-2-methyldec-9-enoic acid Chemical compound OC(=O)C(N)(C)CCCCCCC=C JWZFECWHKQLRGK-UHFFFAOYSA-N 0.000 description 1
- AERCCJGORROTKW-UHFFFAOYSA-N 2-amino-2-methylhept-6-enoic acid Chemical compound OC(=O)C(N)(C)CCCC=C AERCCJGORROTKW-UHFFFAOYSA-N 0.000 description 1
- JDDWRLPTKIOUOF-UHFFFAOYSA-N 9h-fluoren-9-ylmethyl n-[[4-[2-[bis(4-methylphenyl)methylamino]-2-oxoethoxy]phenyl]-(2,4-dimethoxyphenyl)methyl]carbamate Chemical compound COC1=CC(OC)=CC=C1C(C=1C=CC(OCC(=O)NC(C=2C=CC(C)=CC=2)C=2C=CC(C)=CC=2)=CC=1)NC(=O)OCC1C2=CC=CC=C2C2=CC=CC=C21 JDDWRLPTKIOUOF-UHFFFAOYSA-N 0.000 description 1
- 102000044503 Antimicrobial Peptides Human genes 0.000 description 1
- 108700042778 Antimicrobial Peptides Proteins 0.000 description 1
- 101710081722 Antitrypsin Proteins 0.000 description 1
- 241001574543 Boana Species 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- 206010061818 Disease progression Diseases 0.000 description 1
- 241001522957 Enterococcus casseliflavus Species 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 241000192125 Firmicutes Species 0.000 description 1
- -1 Fmoc amino acid Chemical class 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 241001313840 Hydrophylax gracilis Species 0.000 description 1
- 241000588747 Klebsiella pneumoniae Species 0.000 description 1
- 239000012880 LB liquid culture medium Substances 0.000 description 1
- 206010057190 Respiratory tract infections Diseases 0.000 description 1
- 241000191963 Staphylococcus epidermidis Species 0.000 description 1
- RHQDFWAXVIIEBN-UHFFFAOYSA-N Trifluoroethanol Chemical compound OCC(F)(F)F RHQDFWAXVIIEBN-UHFFFAOYSA-N 0.000 description 1
- UIRFWOOIGULZQN-UHFFFAOYSA-L [Ru](Cl)Cl.C1(=CC=CC=C1)C([P](C1CCCCC1)(C1CCCCC1)C1CCCCC1)[P](C1CCCCC1)(C1CCCCC1)C1CCCCC1 Chemical compound [Ru](Cl)Cl.C1(=CC=CC=C1)C([P](C1CCCCC1)(C1CCCCC1)C1CCCCC1)[P](C1CCCCC1)(C1CCCCC1)C1CCCCC1 UIRFWOOIGULZQN-UHFFFAOYSA-L 0.000 description 1
- 230000000397 acetylating effect Effects 0.000 description 1
- 229920003180 amino resin Polymers 0.000 description 1
- 230000000845 anti-microbial effect Effects 0.000 description 1
- 230000001475 anti-trypsic effect Effects 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
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- 238000002983 circular dichroism Methods 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
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- 229940125904 compound 1 Drugs 0.000 description 1
- 229940125782 compound 2 Drugs 0.000 description 1
- 229940126214 compound 3 Drugs 0.000 description 1
- 238000009833 condensation Methods 0.000 description 1
- 230000005494 condensation Effects 0.000 description 1
- 239000012043 crude product Substances 0.000 description 1
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- 238000010828 elution Methods 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 229940088598 enzyme Drugs 0.000 description 1
- IDGUHHHQCWSQLU-UHFFFAOYSA-N ethanol;hydrate Chemical compound O.CCO IDGUHHHQCWSQLU-UHFFFAOYSA-N 0.000 description 1
- LCFXLZAXGXOXAP-QPJJXVBHSA-N ethyl (2e)-2-cyano-2-hydroxyiminoacetate Chemical compound CCOC(=O)C(=N\O)\C#N LCFXLZAXGXOXAP-QPJJXVBHSA-N 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
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- 239000000463 material Substances 0.000 description 1
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- 230000001717 pathogenic effect Effects 0.000 description 1
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- 239000010453 quartz Substances 0.000 description 1
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- 238000012216 screening Methods 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N silicon dioxide Inorganic materials O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
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- ZGYICYBLPGRURT-UHFFFAOYSA-N tri(propan-2-yl)silicon Chemical compound CC(C)[Si](C(C)C)C(C)C ZGYICYBLPGRURT-UHFFFAOYSA-N 0.000 description 1
- 239000002753 trypsin inhibitor Substances 0.000 description 1
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Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Peptides Or Proteins (AREA)
Abstract
The invention belongs to the field of polypeptide medicaments, and in particular relates to a staple peptide and a pharmaceutical application thereof. The invention relates to a template polypeptide Figainin2: ac-FLGAILKIGHALAKTVLPMVTNAFKPKQ-NH 2 Modification of amino acid sequence, while maintaining key amino acid residues, the amino acid positions i, i+4 are respectively modified with S 5 The target stapling peptide is obtained by replacing the original amino acid. The stapler peptide prepared by the invention has higher alpha helicity than linear peptide, has stability to trypsin and chymotrypsin, and has inhibitory activity to Acinetobacter baumannii, pseudomonas aeruginosa, staphylococcus aureus and escherichia coliHas potential application value in the treatment of related diseases such as clinical drug-resistant bacteria infection and the like.
Description
Technical Field
The invention belongs to the field of polypeptide medicaments, and in particular relates to a stapler peptide capable of improving alpha helicity, enzymolysis resistance stability and gram positive and gram negative bacteria resistance activity on the basis of a template polypeptide and pharmaceutical application thereof.
Background
Bacterial infections are a major health threat to humans, which result in a wide variety of diseases. Bacteria invade human tissues and multiply, and cause a series of pathophysiological reactions, thereby causing serious harm to human health. Such bacterial infections can cause various degrees of disease progression throughout the human body including, but not limited to, respiratory tract infections, urinary system infections, digestive system infections, and the like. These diseases can cause serious discomfort and health problems to the human body and in some cases can be life threatening. Meanwhile, the abuse of antibiotics leads to the emergence of drug-resistant strains, and infectious diseases caused by the drug-resistant strains have become a serious threat to public health worldwide. Clinically resistant strains such as staphylococcus aureus, pseudomonas aeruginosa, acinetobacter baumannii and the like can form a layer of biological film on the surface of the strain, and the tolerance of bacteria wrapped by the biological film to conventional antibiotics is 10-1000 times that of other bacteria, so that clinically available antibiotics have certain limitation in resisting the resistant bacteria. In recent years, development of antibacterial drugs has been slow, and part of the antibiotic drugs on the market have developed drug-resistant bacteria in a short time.
Figainin2 is a novel antimicrobial peptide isolated from skin secretions of the Rana gracilis (Boana ranices). In 2020, santana group analysis studied the antibacterial peptide Figainin2 (F2-1), which contains 28 residues and has an alpha helix structure. Figainin2 is relatively sensitive to gram-positive bacteria such as staphylococcus epidermidis and enterococcus casseliflavus (mic=4 μm), and has antibacterial effect against gram-negative bacteria such as escherichia coli and klebsiella pneumoniae (mic=8 μm). Figainin2 has the following disadvantages: (1) The structure is unstable and is easy to be hydrolyzed, so that the medicinal efficacy is reduced; (2) The antibacterial activity is not good enough, and particularly for pseudomonas aeruginosa, the pseudomonas aeruginosa can be inhibited by higher concentration, and the development of the pseudomonas aeruginosa serving as a medicament is influenced by the higher concentration, which means that the clinical dosage is large.
Disclosure of Invention
The invention aims at overcoming the defects in the prior art and provides a staple peptide with high drug-resistant bacteria activity and high stability.
It is a further object of the present invention to provide the use of the stapling peptides.
In order to achieve the first object, the invention adopts the following technical scheme:
a staple peptide selected from one of the following:
a) Ac-FLGAILKIGHALAKTVLPMVTNAFKPKQ-NH 2 Is a peptide chain template in which amino acid residues 1F and 5I are S 5 Replacing and cyclization;
b) Ac-FLGAILKIGHALAKTVLPMVTNAFKPKQ-NH 2 Is a peptide chain template in which amino acid residues 4A and 8I are S 5 Replacing and cyclization;
c) Ac-FLGAILKIGHALAKTVLPMVTNAFKPKQ-NH 2 Is a peptide chain template in which the amino acid residuesRadicals 5I and 9G are S 5 Replacing and cyclization;
d) Ac-FLGAILKIGHALAKTVLPMVTNAFKPKQ-NH 2 Is a peptide chain template in which amino acid residues 9G and 13A are S 5 Replacing and cyclization;
e) Ac-FLGAILKIGHALAKTVLPMVTNAFKPKQ-NH 2 Is a peptide chain template in which amino acid residues 15T and 19M are S 5 Replacing and cyclization;
f) Ac-FLGAILKIGHALAKTVLPMVTNAFKPKQ-NH 2 Is a peptide chain template in which amino acid residues 16V and 20V are S 5 Replacing and cyclization;
g) Ac-FLGAILKIGHALAKTVLPMVTNAFKPKQ-NH 2 Is a peptide chain template in which amino acid residues 18P and 22N are S 5 Replacing and cyclization;
h) Ac-FLGAILKIGHALAKTVLPMVTNAFKPKQ-NH 2 Is a peptide chain template in which amino acid residues 19M and 23A are S 5 Replacing and cyclization;
i) Ac-FLGAILKIGHALAKTVLPMVTNAFKPKQ-NH 2 Is a peptide chain template in which amino acid residues 20V and 24F are S 5 Replacing and cyclization;
j) Ac-FLGAILKIGHALAKTVLPMVTNAFKPKQ-NH 2 Is a peptide chain template in which amino acid residues 22N and 26P are S 5 Replacing and cyclization;
k) Ac-FLGAILKIGHALAKTVLPMVTNAFKPKQ-NH 2 Is a peptide chain template in which amino acid residues 24F and 28Q are S 5 Replacing and cyclization;
the sequences of the template polypeptides and engineered staple peptides of the invention are shown in the following table:
TABLE 1 sequences of template polypeptides and engineered staple peptides of the invention
The application of the staple peptide in preparing medicines for inhibiting Acinetobacter baumannii, pseudomonas aeruginosa, staphylococcus aureus and escherichia coli.
The use of the above-described stapling peptides for increasing alpha helicity.
The application of the staple peptide in trypsin and chymotrypsin stabilization.
The invention has the advantages that:
1. the invention provides a series of novel staple peptides, which are obtained by structurally modifying Figainin-2 (F2-1) so as to have higher alpha helicity, enzymolysis stability of antitrypsin and chymotrypsin and drug-resistant bacteria activity.
2. In order to improve the stability and the drug-resistant bacteria activity of the polypeptide, the patent reforms F2-1 by stapling, reforms the i, i+4 site on the basis of keeping the key residue, and uses S at the non-key residue position of the peptide chain 5 The original i and i+4 amino acids are replaced, and the structure-stable stapling peptide is obtained after cyclization. The patent designs 11 pieces of staple peptides (F2-F2-12) together, and results show that the enzymatic stability of the template polypeptide can be improved by the screened staple peptides. Protease-mediated protease stabilization experiments showed that the template peptide F2-1 was completely degraded after 1.5h of trypsin exposure. At the same time, at least 30.3% of the stapled peptide F2-12 remains intact. In contrast, 10% of the stapled peptide F2-12 remained intact after 2.5 hours of exposure to the same trypsin. Chymotrypsin-mediated protease stabilization experiments indicate that F2-1 is completely degraded after 2.5h, while at least 12.3% of the stapled peptide F2-12 remains intact. F2-12 has a remarkable stability against trypsin and chymotrypsin compared to the template peptide F2-1. One possible reason is that the locked conformation of F2-12 limits the contact between the protease and the degradation site. Meanwhile, the polypeptide obtained by screening improves the antibacterial activity, and can produce inhibitory forward regulation and control on harmful drug-resistant bacteria. Wherein, the antibacterial effect of F2-10 and F2-12 on staphylococcus aureus is obviously improved, and the inhibitory effect of F2-12 on acinetobacter baumannii is obviously enhanced.
Drawings
FIG. 1 is a schematic representation of the amino acid sequence and characterization of F2-1, F2-1 being purified by HPLC (purification conditions: 10% -60% CH) 3 CN,55 min), F2-1 was obtained with a separation rate of 24.4%. Wherein A is the amino acid sequence of F2-1, B is the HPLC profile of F2-1, analytical column: c18, gradient: 10% -90% CH 3 CN,25 min). C is a mass spectrum of F2-1. FIG. 2 is a schematic representation of the amino acid sequence and characterization of F2-2, F2-2 being purified by HPLC (purification conditions: 10% -55% CH) 3 CN,55 min), F2-2 was obtained with a separation of 14.7%. Wherein A is the amino acid sequence of F2-2, B is the HPLC profile of F2-2, analytical column: c18, gradient: 10% -90% CH 3 CN,25 min). C is a mass spectrum of F2-2. FIG. 3 is a schematic representation of the amino acid sequence and characterization of F2-3, F2-3 being purified by HPLC (purification conditions: 10% -60% CH) 3 CN,55 min), F2-3 was obtained with a separation of 14.6%. Wherein A is the amino acid sequence of F2-3, B is the HPLC profile of F2-3, analytical column: c18, gradient: 10% -90% CH 3 CN 25 min). C is a mass spectrum of F2-3.
FIG. 4 is a schematic representation of the amino acid sequence of F2-4 and its characterization, purification of F2-4 by HPLC (purification conditions: 10% -60% CH 3 CN,55 min), F2-4 was obtained with a separation of 19.6%. Wherein A is the amino acid sequence of F2-4, B is the HPLC profile of F2-4, analytical column: c18, gradient: 10% -90% CH 3 CN,25 min). C is a mass spectrum of F2-4. FIG. 5 is a schematic representation of the amino acid sequence and characterization of F2-5, F2-5 being purified by HPLC (purification conditions: 10% -55% CH) 3 CN,55 min), F2-5 was obtained. Wherein A is the amino acid sequence of F2-5, B is the HPLC profile of F2-5, analytical column: c18, gradient: 10% -90% CH 3 CN,25min. C is a mass spectrum of F2-5.
FIG. 6 is a schematic representation of the amino acid sequence of F2-6 and its characterization, purification of F2-6 by HPLC (purification conditions: 10% -55% CH 3 CN,55 min) to give F2-6. Wherein A is the amino acid sequence of F2-6, B is the HPLC profile of F2-6, analytical column: c18, gradient: 10% -90% CH 3 CN,25min. C is a mass spectrum of F2-6.
FIG. 7 shows the amino acid sequence of F2-7Intention and its characterization profile, F2-7 was purified by HPLC (purification conditions: 10% -55% CH 3 CN,55 min) to give F2-7. Wherein A is the amino acid sequence of F2-7, B is the HPLC profile of F2-7, analytical column: c18, gradient: 10% -90% CH 3 CN,25min. C is a mass spectrum of F2-7.
FIG. 8 is a schematic representation of the amino acid sequence and characterization of F2-8, F2-8 being purified by HPLC (purification conditions: 10% -55% CH) 3 CN,55 min), F2-8 was obtained. Wherein A is the amino acid sequence of F2-8, B is the HPLC profile of F2-8, analytical column: c18, gradient: 10% -90% CH 3 CN,25min. C is a mass spectrum of F2-8.
FIG. 9 is a schematic representation of the amino acid sequence of F2-9 and its characterization, wherein F2-9 is purified by HPLC (purification conditions: 10% -55% CH 3 CN,55 min) to give F2-9. Wherein A is the amino acid sequence of F2-9, B is the HPLC profile of F2-9, analytical column: c18, gradient: 10% -90% CH 3 CN,25min. C is a mass spectrum of F2-6.
FIG. 10 is a schematic representation of the amino acid sequence and characterization of F2-10, F2-10 being purified by HPLC (purification conditions: 10% -55% CH) 3 CN,55 min), F2-10 was obtained. Wherein A is the amino acid sequence of F2-10, B is the HPLC profile of F2-10, analytical column: c18, gradient: 10% -90% CH 3 CN,25min. C is a mass spectrum of F2-10.
FIG. 11 is a schematic representation of the amino acid sequence of F2-11 and its characterization, purification of F2-11 by HPLC (purification conditions: 10% -55% CH 3 CN,55 min) to give F2-11. Wherein A is the amino acid sequence of F2-11, B is the HPLC profile of F2-11, analytical column: c18, gradient: 10% -90% CH 3 CN,25min. C is a mass spectrum of F2-11.
FIG. 12 is a schematic representation of the amino acid sequence and characterization of F2-12, F2-12 being purified by HPLC (purification conditions: 10% -55% CH 3 CN,55 min), F2-12 was obtained. Wherein A is the amino acid sequence of F2-12, B is the HPLC profile of F2-12, analytical column: c18, gradient: 10% -90% CH 3 CN,25min. C is a mass spectrum of F2-12.
FIG. 13 shows the proteolytic stability of template peptides F2-1 and staple peptides F2-12 in trypsin solution and chymotrypsin solution. Wherein A is the proteolytic stability of the polypeptide in trypsin solution; b is the proteolytic stability of the polypeptide in chymotrypsin solution.
Detailed Description
The present application will be further described with reference to the drawings and detailed description so as to be more readily apparent to those skilled in the art, but such examples are intended to illustrate the invention and not to limit the scope of the invention, i.e. the examples described are only some, but not all, of the examples of the invention.
Thus, the following detailed description of certain embodiments of the present invention is not intended to limit the scope of the invention, as claimed, but is merely a selection of embodiments of the invention. All other embodiments, which can be made by a person skilled in the art without making any inventive effort, are intended to be within the scope of the present invention.
In the present invention, the abbreviations involved are explained as follows:
fmoc: fluorenylmethoxycarbonyl;
DIEA: n, N-diisopropylethylamine
Ac 2 O: acetic anhydride
DCM: dichloromethane (dichloromethane)
DCE:1, 2-dichloroethane
DMF: n, N-dimethylformamide
Oxyme：Ethyl Cyanoglyoxylate-2-Oxime
DIC: n, N-diisopropylcarbodiimide
S 5 ：2-amino-2-methylhept-6-enoic acid
R 8 ：2-amino-2-methyldec-9-enoic acid
TFA: trifluoroacetic acid
TIPS: triisopropylsilane
Grubbs i: phenyl methylene bis (tricyclohexylphosphorus) ruthenium dichloride
MS: mass spectrometry
HR-Q-TOF-MS: high resolution matrix assisted laser desorption ionization time-of-flight mass spectrometry.
The experimental materials involved in the embodiment of the invention are derived from the following sources: fmoc-amino acid, rink amide MBHA resin was purchased from Nanking Synthesis Co., ltd; NMP, DIC, oxyme, TFA, acetonitrile purchased from an exploration platform; DMF, anhydrous diethyl ether, DCM, DCE, piperidine and phenol are all analytically pure and purchased from Shanghai Co., ltd
The invention is based on template polypeptide F2-1:Ac-FLGAILKIGHALAKTVLPMVTNAFKPKQ-NH 2 The amino acid sequence was designed and 11 staples were synthesized. The structure and molecular weight of the stapled peptides are shown in table 1.
Example 1 preparation of Figainin2 (F2-1) based staple peptides
1. Synthesis of staple peptides
(1) Preparation of Compound 1
Adding 600mg of amino resin into a solid phase synthesis reaction tube, soaking in DCM for 30min to fully swell the resin, and pumping for later use.
20% piperidine-DMF solution was added to the resin and the Fmoc protecting groups on the resin were removed by shaking at 35℃for 5min, followed by subsequent washing with DMF, DCM, DMF to remove the piperidine-DMF solution from the resin.
(2) Preparation of Compound 2
The first amino acid (1 mmol), oxyme (1 mmol) and DIC (200 μl) in the sequence were dissolved in 7mL DMF and added to the solid phase synthesis reaction tube and the resin was shaken at 50 ℃ for 60min, followed by washing the resin with DMF, DCM, DMF.
(3) Preparation of Compound 3
Repeating the steps (1) and (2), sequentially dissolving Fmoc amino acid (1 mmol), oxyme (1 mmol) and DIC (200 mu L) in 7mL of DMF according to the polypeptide sequence, adding the solution to the resin, and carrying out oscillation reaction at 50 ℃ for 60min, and repeating the processes of Fmoc protection removal, amino acid condensation and Fmoc protection removal until all amino acids are condensed. After removal of Fmoc protecting group from the last amino acid, ac is added 2 O: DIEA: DMF (1:1:8) mixture was shaken at 37℃for 30min, pumped down, added with acetylating reagent again, reacted for 30min, washed the resin with DMF, DCM, DMF in turn, and dried by pumping vacuum through an oil pump.
(4) Preparation of Compound 4
After the resin was completely dried, 1, 2-dichloroethane solution (10 mL) containing Grubbs I (100 mg) was added thereto, and the reaction was carried out by shaking at 37℃and then the resin was washed with DMF, DCM, DMF in sequence after the completion of the reaction, and the resin was dried by pumping under vacuum.
(5) Preparation of target Compounds
The resin was first washed and drained and TFA, phenol, H were added 2 Tips=88.75:0.5:0.5:0.25 (V/V) 20ml, shaking 3h at 37 ℃, filtering and collecting the filtrate. And (3) precipitating and centrifuging the mixture by using glacial ethyl ether, removing supernatant, and naturally volatilizing the supernatant under a fume hood to obtain a crude polypeptide sample.
2. Purification of stapled peptide samples
Dissolving the crude polypeptide with a mixed solvent of acetonitrile and water, and purifying by reverse phase preparative RP-HPLC to obtain a purified pure product of the staple peptide. The separation conditions were as follows:
instrument: the Shimadzu LC-20A reversed phase high performance liquid chromatograph;
chromatographic column: ultimateXB-C18, 21.2X250 mm,5 μm;
mobile phase: mobile phase a was acetonitrile solution with a volume fraction of 0.1% tfa, and mobile phase B was aqueous solution with a volume fraction of 0.1% tfa;
the steps and parameters are as follows: 90% -50% of B,40min, the flow rate is 8mL/min, and the detection wavelengths are 214nm and 254nm.
Example 2 identification and Structure analysis of the product
The product from step 2 of example 1 was identified by reverse phase HPLC and analyzed for structure by HR-Q-TOF-MS, the chromatographic mobile phase being acetonitrile and water. Mobile phase A is acetonitrile solution with volume fraction of 0.1% TFA, mobile phase B is water solution with volume fraction of 0.1% TFA, gradient elution is carried out for 90% -10% B,25min; the flow rate is 1.0 mL-min -1 The method comprises the steps of carrying out a first treatment on the surface of the Detection wavelengths were 214nm and 254nm. The time of the main peak of the obtained product is consistent with that of the crude product, and the purity of the staple peptide prepared by the method>95% mass spectrometry results are shown. The structure of the resulting stapled peptides is shown in Table 1.
TABLE 1 sequences of template polypeptides and engineered staple peptides of the invention
EXAMPLE 3 experiment of the inventive staple peptide against gram-Positive and gram-negative bacteria
In vitro anti-drug-resistant bacteria test: preparing a solid LB culture medium, plating the solid LB culture medium after autoclaving, and preparing an LB liquid culture medium for later use in a refrigerator at 4 ℃. Coating the bacterial liquid on a solid LB culture medium, and culturing the bacterial liquid in an incubator at 37 ℃ in an inverted way overnight; taking a monoclonal, adding the monoclonal into 3mL of liquid LB culture medium, culturing for 6 hours at 37 ℃ and 220rpm in a constant-temperature shaking table, and enabling bacteria to grow to a logarithmic phase; taking 1mL of bacterial liquid, centrifuging at 4000rpm for 5min, discarding supernatant, adding PBS, and adjusting bacterial liquid concentration to 2×10 by OD value 6 CFU/mL. Antibacterial peptides with different concentrations are added into a 96-well plate, bacterial liquid is added into the 96-well plate at the same time, the bacterial liquid is cultured for 8 hours at 37 ℃, the enzyme label instrument adopts 595nm for detection, the detection is repeated three times, and MIC values are statistically analyzed.
The results are shown in Table 2.
TABLE 2 results of experiment of the inventive staple peptides against gram-positive and gram-negative bacteria
The results in Table 2 show that the stapled peptides of the invention can improve the drug-resistant activity of the template polypeptide, wherein the F2-12 drug-resistant effect is most prominent. The embodiment shows that the modified staple peptide based on the Figainin2 (F2-1) is successfully prepared, and the in-vitro bacteriostasis experiment proves that the synthesized staple peptide can inhibit the growth and reproduction of pathogenic drug-resistant bacteria, and has the application prospect of being developed into novel antibacterial drugs.
EXAMPLE 4 round dichroism experiments with the stapler peptides of the invention
The linear peptides F2-1 and the stapled peptides F2-2 to F2-12 were precisely weighed 1mg and dissolved in water and trifluoroethanol (1:1) solutions, respectively, to a final concentration of 50mM. Characterization was performed using circular dichroism JASCO J-1500 and a 1mm quartz cuvette at room temperature. The following experimental parameters were measured: wavelength, 190-260nm; quick speedDegree, 20nm.min -1 The method comprises the steps of carrying out a first treatment on the surface of the Bandwidth, 1nm. For the alpha-helical structure, there is one positive band around 192nm and two negative bands at 222nm and 208 nm. Calculating helicity of each peptide according to ellipticity of spectrum of peptide at 222nm and amino acid number in peptide sequence by using equation
The results are shown in Table 3.
TABLE 3 results of the alpha helicity experiments of the inventive staple peptides
The results in Table 3 show that the stapled peptides of the invention can increase the alpha-helicity of the template polypeptide. The above examples show that the modified staple peptide based on the Figainin2 (F2-1) is successfully prepared, and most of the staple peptides have higher helicity than the linear peptide F2-1 through round dichroism analysis experiments, so that the staple locking modified by the staple peptide strategy plays a certain role in strengthening the peptide chain.
EXAMPLE 5 stability test of the inventive staple peptides
To evaluate the protease stability of the peptides, the peptides F2-12 and F2-1 were selected for their highest antimicrobial activity and their susceptibility to trypsin and chymotrypsin degradation was measured. Trypsin and chymotrypsin were dissolved separately in PBS buffer at a final concentration of 10ng/mL. The peptide solution (50. Mu.L) was then incubated with trypsin solution and chymotrypsin (1950. Mu.L) at room temperature. 60. Mu.L of the digested mixture was taken at 0, 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0h, respectively, and then quenched with 20. Mu.L of hydrochloric acid (1M). The peptide solutions were then assayed for various times by High Performance Liquid Chromatography (HPLC) to determine the degradation rate of the protease at 214 nm.
The results are shown in FIG. 13.
The result shows that the stapler peptide can improve the enzymolysis stability of the template polypeptide. Protease-mediated protease stabilization experiments showed that the template peptide F2-1 was completely degraded after 1.5h of trypsin exposure. At the same time, at least 30.3% of the stapled peptide F2-12 remains intact. In contrast, 10% of the stapled peptide F2-12 remained intact after 2.5 hours of exposure to the same trypsin. Chymotrypsin-mediated protease stabilization experiments indicate that F2-1 is completely degraded after 2.5h, while at least 12.3% of the stapled peptide F2-12 remains intact. F2-12 has a remarkable stability against trypsin and chymotrypsin compared to the template peptide F2-1. One possible reason is that the locked conformation of F2-12 limits the contact between the protease and the degradation site.
The foregoing is merely a preferred embodiment of the present invention, and it should be noted that modifications and additions may be made to those skilled in the art without departing from the method of the present invention, which modifications and additions are also to be considered as within the scope of the present invention.
Claims (5)
1. A staple peptide, wherein the staple peptide is selected from one of the following:
a) Ac-FLGAILKIGHALAKTVLPMVTNAFKPKQ-NH 2 Is a peptide chain template in which amino acid residues 1F and 5I are S 5 Replacing and cyclization;
b) Ac-FLGAILKIGHALAKTVLPMVTNAFKPKQ-NH 2 Is a peptide chain template in which amino acid residues 4A and 8I are S 5 Replacing and cyclization;
c) Ac-FLGAILKIGHALAKTVLPMVTNAFKPKQ-NH 2 Is a peptide chain template in which amino acid residues 5I and 9G are S 5 Replacing and cyclization;
d) Ac-FLGAILKIGHALAKTVLPMVTNAFKPKQ-NH 2 Is a peptide chain template in which amino acid residues 9G and 13A are S 5 Replacing and cyclization;
e) Ac-FLGAILKIGHALAKTVLPMVTNAFKPKQ-NH 2 Is a peptide chain template in which amino acid residues 15T and 19M are S 5 Replacing and cyclization;
f) Ac-FLGAILKIGHALAKTVLPMVTNAFKPKQ-NH 2 Is a peptide chain template in which amino acid residues 16V and 20V are S 5 Replacing and cyclization;
g) Ac-FLGAILKIGHALAKTVLPMVTNAFKPKQ-NH 2 Is a peptide chain template in which amino acid residues 18P and 22N are S 5 Replacing and cyclization;
h) Ac-FLGAILKIGHALAKTVLPMVTNAFKPKQ-NH 2 Is a peptideChain templates in which amino acid residues 19M and 23A are S 5 Replacing and cyclization;
i) Ac-FLGAILKIGHALAKTVLPMVTNAFKPKQ-NH 2 Is a peptide chain template in which amino acid residues 20V and 24F are S 5 Replacing and cyclization;
j) Ac-FLGAILKIGHALAKTVLPMVTNAFKPKQ-NH 2 Is a peptide chain template in which amino acid residues 22N and 26P are S 5 Replacing and cyclization;
k) Ac-FLGAILKIGHALAKTVLPMVTNAFKPKQ-NH 2 Is a peptide chain template in which amino acid residues 24F and 28Q are S 5 And (5) replacing and cyclization.
2. Use of a staple peptide according to claim 1 for the preparation of an antibacterial medicament.
3. The staple peptide of claim 2 wherein the bacterium is acinetobacter baumannii, pseudomonas aeruginosa, staphylococcus aureus or escherichia coli.
4. Use of a staple peptide according to claim 1 in a polypeptide secondary structure analysis.
5. Use of the stapled peptide of claim 1 for trypsin and chymotrypsin stability.
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| WO2022237853A1 (en) * | 2021-05-12 | 2022-11-17 | 香港理工大学深圳研究院 | Beclin1-targeted stapled peptide and pharmaceutical composition |
| CN116655766A (en) * | 2023-04-23 | 2023-08-29 | 山东第一医科大学(山东省医学科学院) | Staple peptide and preparation method and application thereof |
| CN116693626A (en) * | 2022-11-18 | 2023-09-05 | 潍坊医学院附属医院 | Staple peptides and uses thereof and methods for expanding stem cells in vitro |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2022237853A1 (en) * | 2021-05-12 | 2022-11-17 | 香港理工大学深圳研究院 | Beclin1-targeted stapled peptide and pharmaceutical composition |
| CN116693626A (en) * | 2022-11-18 | 2023-09-05 | 潍坊医学院附属医院 | Staple peptides and uses thereof and methods for expanding stem cells in vitro |
| CN116655766A (en) * | 2023-04-23 | 2023-08-29 | 山东第一医科大学(山东省医学科学院) | Staple peptide and preparation method and application thereof |
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| Title |
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| CARLOS JOSÉ CORREIA CARLOS JOSÉ CORREIA SANTANA ET AL: "Biological Properties of a Novel Multifunctional Host Defense Peptide from the Skin Secretion of the Chaco Tree Frog, Boana raniceps", BIOMOLECULES, vol. 10, no. 5, 20 May 2020 (2020-05-20), pages 1 * |
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