CN117756905B - Staple peptide and pharmaceutical application thereof - Google Patents
Staple peptide and pharmaceutical application thereof Download PDFInfo
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- CN117756905B CN117756905B CN202311782032.7A CN202311782032A CN117756905B CN 117756905 B CN117756905 B CN 117756905B CN 202311782032 A CN202311782032 A CN 202311782032A CN 117756905 B CN117756905 B CN 117756905B
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- 241000894006 Bacteria Species 0.000 claims abstract description 19
- 239000003814 drug Substances 0.000 claims abstract description 17
- 125000000539 amino acid group Chemical group 0.000 claims abstract description 13
- 241000588626 Acinetobacter baumannii Species 0.000 claims abstract description 6
- 241000191967 Staphylococcus aureus Species 0.000 claims abstract description 6
- 230000000844 anti-bacterial effect Effects 0.000 claims description 6
- 238000004519 manufacturing process Methods 0.000 claims 1
- 102000004196 processed proteins & peptides Human genes 0.000 abstract description 49
- 125000003275 alpha amino acid group Chemical group 0.000 abstract description 27
- 229920001184 polypeptide Polymers 0.000 abstract description 19
- 108090000631 Trypsin Proteins 0.000 abstract description 14
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- 239000012588 trypsin Substances 0.000 abstract description 14
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- 238000007792 addition Methods 0.000 description 2
- 229940124350 antibacterial drug Drugs 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
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- 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
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- 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
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- 241001574543 Boana Species 0.000 description 1
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- 241000192125 Firmicutes Species 0.000 description 1
- -1 Fmoc amino acid Chemical class 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 241000588747 Klebsiella pneumoniae Species 0.000 description 1
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- 241000191963 Staphylococcus epidermidis Species 0.000 description 1
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- 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
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- 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
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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
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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
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- 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. According to the invention, modification is carried out according to the amino acid sequence Ac-FLGAILKIGHALAKTVLPMVTNAFKPKQ-NH 2 of the template polypeptide Figainin, and S 5 is used for replacing original amino acids at the i, i+4 amino acid positions on the basis of keeping key amino acid residues, so as to obtain the target stapling peptide. The prepared staple peptide has higher alpha helicity than linear peptide, has stability to trypsin and chymotrypsin, has inhibitory activity to Acinetobacter baumannii, pseudomonas aeruginosa, staphylococcus aureus and escherichia coli, and has potential application value in treating 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.
Figainin 2A novel antibacterial peptide is isolated from skin secretion of Rana chalcogramma (Boana raniceps). In 2020, santana group analysis studied antibacterial peptide Figainin (F2-1), which contains 28 residues and has an alpha helix structure. Figainin 2A is relatively sensitive to gram-positive bacteria such as Staphylococcus epidermidis and enterococcus casseliflavus (MIC=4μm), and has antibacterial effect on gram-negative bacteria such as Escherichia coli and Klebsiella pneumoniae (MIC=8μm). Figainin 2 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 used as a peptide chain template, wherein amino acid residues 1F and 5I are replaced by S 5 and are cyclized;
b) Ac-FLGAILKIGHALAKTVLPMVTNAFKPKQ-NH 2 is used as a peptide chain template, wherein amino acid residues 4A and 8I are replaced by S 5 and are cyclized;
c) Ac-FLGAILKIGHALAKTVLPMVTNAFKPKQ-NH 2 is used as a peptide chain template, wherein amino acid residues 5I and 9G are replaced by S 5 and are cyclized;
d) Ac-FLGAILKIGHALAKTVLPMVTNAFKPKQ-NH 2 is used as a peptide chain template, wherein amino acid residues 9G and 13A are replaced by S 5 and are cyclized;
e) Ac-FLGAILKIGHALAKTVLPMVTNAFKPKQ-NH 2 is used as a peptide chain template, wherein the amino acid residues 15T and 19M are replaced by S 5 and are cyclized;
f) Ac-FLGAILKIGHALAKTVLPMVTNAFKPKQ-NH 2 is used as a peptide chain template, wherein amino acid residues 16V and 20V are replaced by S 5 and are cyclized;
g) Ac-FLGAILKIGHALAKTVLPMVTNAFKPKQ-NH 2 is used as a peptide chain template, wherein amino acid residues 18P and 22N are replaced by S 5 and are cyclized;
h) Ac-FLGAILKIGHALAKTVLPMVTNAFKPKQ-NH 2 is used as a peptide chain template, wherein amino acid residues 19M and 23A are replaced by S 5 and are cyclized;
i) Ac-FLGAILKIGHALAKTVLPMVTNAFKPKQ-NH 2 is used as a peptide chain template, wherein amino acid residues 20V and 24F are replaced by S 5 and are cyclized;
j) Ac-FLGAILKIGHALAKTVLPMVTNAFKPKQ-NH 2 is used as a peptide chain template, wherein amino acid residues 22N and 26P are replaced by S 5 and are cyclized;
k) Ac-FLGAILKIGHALAKTVLPMVTNAFKPKQ-NH 2 is used as a peptide chain template, wherein amino acid residues 24F and 28Q are replaced by S 5 and are cyclized;
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 structural modification of Figainin-2 (F2-1), so that the novel staple peptides have higher alpha helicity, enzymolysis stability of antitrypsin and chymotrypsin and drug-resistant bacteria activity, and pharmacological experiments show that the novel staple peptides have higher alpha helicity than linear peptides, have stability to trypsin and chymotrypsin, have inhibitory activity to Acinetobacter baumannii, pseudomonas aeruginosa, staphylococcus aureus and escherichia coli, and have potential application value in the treatment of related diseases such as clinical drug-resistant bacteria infection.
2. In order to improve the stability and the drug-resistant bacteria activity of the polypeptide, the patent carries out stapling transformation on F2-1, and on the basis of keeping key residues, the transformation is carried out on i, i+4 sites, S 5 is used for replacing the original i and i+4 site amino acids at the non-key residue position of a peptide chain, and the stapling peptide with stable structure 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 diagram of the amino acid sequence of F2-1 and its characterization, wherein F2-1 is purified by HPLC (purification conditions: 10% -60% CH 3 CN,55 min), to give F2-1 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 diagram of the amino acid sequence of F2-2 and its characterization, wherein F2-2 is purified by HPLC (purification conditions: 10% -55% CH 3 CN,55 min), to give F2-2 with a separation rate 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 diagram of the amino acid sequence of F2-3 and its characterization, wherein F2-3 is purified by HPLC (purification conditions: 10% -60% CH 3 CN,55 min), to give F2-3 with a separation rate 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 diagram of the amino acid sequence of F2-4 and its characterization, wherein F2-4 is purified by HPLC (purification conditions: 10% -60% CH 3 CN,55 min), to give F2-4 with a separation rate 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 of F2-5 and its characterization, and F2-5 is purified by HPLC (purification conditions: 10% -55% CH 3 CN,55 min) to give F2-5. Wherein A is the amino acid sequence of F2-5, B is the HPLC profile of F2-5, analytical column: c18, gradient: 10% -90% of CH 3 CN for 25min. C is a mass spectrum of F2-5.
FIG. 6 is a schematic diagram of the amino acid sequence of F2-6 and its characterization, and F2-6 is purified 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% of CH 3 CN for 25min. C is a mass spectrum of F2-6.
FIG. 7 is a schematic diagram of the amino acid sequence of F2-7 and its characterization, and F2-7 is 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% of CH 3 CN for 25min. C is a mass spectrum of F2-7.
FIG. 8 is a schematic representation of the amino acid sequence of F2-8 and its characterization, and F2-8 is purified by HPLC (purification conditions: 10% -55% CH 3 CN,55 min) to give F2-8. Wherein A is the amino acid sequence of F2-8, B is the HPLC profile of F2-8, analytical column: c18, gradient: 10% -90% of CH 3 CN for 25min. C is a mass spectrum of F2-8.
FIG. 9 is a schematic diagram of the amino acid sequence of F2-9 and its characterization, and 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% of CH 3 CN for 25min. C is a mass spectrum of F2-6.
FIG. 10 is a schematic diagram of the amino acid sequence of F2-10 and its characterization, and F2-10 is purified by HPLC (purification conditions: 10% -55% CH 3 CN,55 min) to give F2-10. Wherein A is the amino acid sequence of F2-10, B is the HPLC profile of F2-10, analytical column: c18, gradient: 10% -90% of CH 3 CN for 25min. C is a mass spectrum of F2-10.
FIG. 11 is a schematic diagram of the amino acid sequence of F2-11 and its characterization, and F2-11 is purified 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% of CH 3 CN for 25min. C is a mass spectrum of F2-11.
FIG. 12 is a schematic diagram of the amino acid sequence of F2-12 and its characterization, and F2-12 is purified by HPLC (purification conditions: 10% -55% CH 3 CN,55 min), to give F2-12. Wherein A is the amino acid sequence of F2-12, B is the HPLC profile of F2-12, analytical column: c18, gradient: 10% -90% of CH 3 CN for 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 accompanying drawings and detailed description so that those skilled in the art will better understand the present application, but these examples are intended to illustrate the present application and not to limit the scope of the present application, i.e. the described examples are only some, but not all, examples of the present application.
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
S5：2-amino-2-methylhept-6-enoic acid
R8：2-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 Nanka Synthesis Inc.; NMP, DIC, oxyme, TFA, acetonitrile is 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 designs and synthesizes 11 staple peptides according to the amino acid sequence of the template polypeptide F2-1:Ac-FLGAILKIGHALAKTVLPMVTNAFKPKQ-NH 2. The structure and molecular weight of the stapled peptides are shown in table 1.
Example 1 preparation of Figainin (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 sequential 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. Mu.L) 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 DMF, DCM, DMF washes.
(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 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 removing Fmoc protecting group from the last amino acid, adding Ac 2 O/DIEA/DMF (1:1:8) mixture, oscillating at 37 ℃ for 30min, pumping, adding an acetylating reagent again, reacting for 30min, washing resin with DMF, DCM, DMF in sequence, and vacuumizing and drying the resin by 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 washed with DMF, DCM, DMF parts of the resin 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, TFA: phenol: H 2 O: TIPS=88.75:0.5:0.5:0.25 (V/V/V) 20mL was added, shaken for 3H at 37 ℃, filtered and the filtrate was collected. 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, 21.2X105 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; detection wavelengths were 214nm and 254nm. The time of the peak is consistent with that of the main peak of the crude product, the purity of the staple peptide prepared by the method is more than 95%, and the mass spectrum analysis result is shown in the figure. 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; 1mL of the bacterial liquid is taken, centrifuged at 4000rpm for 5min, the supernatant is discarded, PBS is added, and the bacterial liquid concentration is adjusted to 2X 10 6 CFU/mL by OD value. 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 invention successfully prepares the reconstructed staple peptide based on Figainin (F2-1), and the in vitro bacteriostasis experiment proves that the synthesized staple peptide can inhibit the growth and propagation 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; speed, 20nm.min -1; 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 invention successfully prepares the modified staple peptide based on Figainin (F2-1), and most of the staple peptides have higher helicity than the linear peptide F2-1 through round dichroism analysis experiments, which indicates that the staple locking after the modification of 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 (2)
1. A staple peptide, wherein the staple peptide is:
k) Ac-FLGAILKIGHALAKTVLPMVTNAFKPKQ-NH 2 was used as a peptide chain template in which amino acid residues 24F and 28Q were replaced by S 5 and were cyclized.
2. Use of a staple peptide of claim 1 in the manufacture of an antibacterial medicament; the bacteria are Acinetobacter baumannii or staphylococcus aureus.
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