CN115925988B - Denatured collagen targeted antibacterial peptide and preparation method and application thereof - Google Patents
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Abstract
The application belongs to the technical field of medicines, and discloses a denatured collagen targeted antibacterial peptide, a preparation method and application thereof. The coupling sequence from N end to C end in the denatured collagen targeted antibacterial peptide is (antibacterial peptide) x -branch point-linker-collagen hybrid peptide or collagen hybrid peptide-linker-branch point- (antibacterial peptide) x The method comprises the steps of carrying out a first treatment on the surface of the Wherein x is a positive integer not less than 2; and the x antibacterial peptides are connected in parallel. The denatured collagen targeted antibacterial peptide can target a great amount of damaged and degraded conformationally denatured collagen molecules existing in infected tissues, so that the collagen targeted antibacterial peptide stays in the tissues in an infected environment and continuously plays a role in treating, reduces side effects on normal tissues, and has a wide application prospect in the field of external anti-infection.
Description
Technical Field
The application belongs to the technical field of medicines, and particularly relates to a denatured collagen targeted antibacterial peptide, and a preparation method and application thereof.
Background
Antibacterial peptides (AMPs) are polypeptides which are present in vertebrates, invertebrates and plants, mostly consisting of amino acids with hydrophobic and positive charges, and have a peptide chain length of less than 100 amino acids, and have the ability to kill pathogenic microorganisms such as bacteria, fungi, viruses, etc. in a broad spectrum. Because of limited drug resistance, the antibacterial peptide drug becomes a potential substitute of conventional antibiotics, and has wide application potential in anti-infection treatment. Although the antibacterial peptide is often quite selective for bacteria of procaryotic cells, the dosage of the antibacterial peptide reaching an infected area is only a small part of the total dosage after entering the systemic circulatory system, and meanwhile, the antibacterial peptide can have certain toxic and side effects (including toxicity to digestive tracts, kidneys and the like) on abnormal tissues, so that the total dosage is limited, and the application is limited. Therefore, the general antibacterial agents (antibacterial peptides, etc.) cannot realize the antibacterial effect in situ, efficiently and for a long time in infected tissues, and simultaneously cannot selectively reduce the adverse effects on normal human tissues.
The prior antibacterial drugs have complex application procedures, and the pathogenic bacteria can be used in a targeted manner only by determining the drug sensitivity test; in addition, systemic administration is prone to toxic and side effects and drug resistance. Although antimicrobial peptides have been reported for prophylactic treatment of wounds and infections, there is no specificity or targeting for the site of infection. If the medicine is taken by normal tissues, not only the effective concentration of the medicine is reduced to cause waste, but also certain toxic and side effects can be generated. It is known that when the body is infected, the infection damages the structure of collagen, which is the most important component of human surface tissue, so that a great deal of structurally damaged collagen exists in wound tissue, and the molecular structure is characterized by denatured collagen conformation. Therefore, if the antibacterial drug targeting the denatured collagen can be developed, the antibacterial drug has a huge application prospect for the external treatment of infectious diseases of various tissues such as skin, soft and hard tissues of skeletal muscle system, oral tissues, reproductive systems, eyes, digestive tracts and the like.
Based on the above, the application hopes to research and modify the antibacterial peptide, and endow the antibacterial peptide with the property of target infection injury tissue denatured collagen so as to better play an antibacterial effect.
Disclosure of Invention
The present application aims to solve at least one of the technical problems in the prior art described above. Therefore, the application provides a denatured collagen targeted antibacterial peptide, and a preparation method and application thereof. The denatured collagen targeted antibacterial peptide can target a great amount of damaged and degraded conformational denatured collagen existing in infected tissues, so that the denatured collagen targeted antibacterial peptide stays in the tissues of the infected environment and continuously plays a role in treating, reduces side effects on normal tissues, and has a wide application prospect in the field of external anti-infection.
The application provides a denatured collagen targeted antibacterial peptide, the coupling sequence from the N end to the C end is (antibacterial peptide) x -branch point-linker-collagen hybrid peptide or collagen hybrid peptide-linker-branch point- (antibacterial peptide) x The method comprises the steps of carrying out a first treatment on the surface of the Wherein x is a positive integer not less than 2; and the x antibacterial peptides are connected in parallel.
Because the collagen hybridization peptide (Collagen hybridizing peptide, CHP) can be hybridized with the triple helix of the modified collagen molecule which is in the conformational unwinding of wounds and infectious inflammatory lesions, the collagen hybridization peptide and the antibacterial peptide are coupled, so that the antibacterial peptide can be anchored in tissues in an infectious environment to exert the drug effect for a long time, and the better antibacterial effect is achieved.
However, it has also been found in research and development that if 2 or more antibacterial peptides are linked in tandem at the N-terminus of a collagen hybrid peptide or 4 or more antibacterial peptides are linked in tandem at the C-terminus of a collagen hybrid peptide, the antibacterial performance thereof may be improved, but another important function of the polypeptide, namely, the ability to target denatured collagen and water solubility, may become undesirable, greatly affecting the overall function thereof. Therefore, not all coupling types can lead the antibacterial peptide to have the functions of targeting collagen and realizing better antibacterial performance, and only coupling a plurality of antibacterial peptides (2 or more) with collagen hybrid peptide in parallel can play a good effect.
Preferably, the coupling sequence from N end to C end in the denatured collagen-targeted antibacterial peptide is (antibacterial peptide) x -branch point-linker-collagen hybrid peptide. Compared with the method that 2 or more antibacterial peptides are connected in parallel at the C end of the collagen hybrid peptide, the method has the advantages that the synthesis process of connecting 2 or more antibacterial peptides in parallel at the N end of the collagen hybrid peptide is simpler and more convenient, and is beneficial to practical development and application.
Preferably, the value of x is 2-4.
More preferably, the value of x is 2.
Preferably, the collagen hybridizing peptide is (GfO) n Or (GPO) n N has the values of 6, 7, 8, 9 and 10. Wherein f is 4-fluoroproline, G is glycine, O is hydroxyproline, and P is proline.
More preferably, the collagen hybridizing peptide is (GfO) 9 。(GfO) 9 Is GfOGFOGFOGFOGFOGFOGFOGFOGFOGFOGFOGFO.
Preferably, the antimicrobial peptide is Feleucoin-K3 (identified as FLC) and has an amino acid sequence of FLKLLKKLL. The antibacterial peptide FLC is a reported cationic antibacterial peptide separated from the skin secretion of Bombina orientalis of Asian toad species, has a secondary structure of alpha-helix and has antibacterial activity on escherichia coli, staphylococcus aureus, candida albicans and the like. In embodiments of the application, other types of antimicrobial peptides may be used to make denatured collagen-targeted antimicrobial peptides.
Preferably, the branch point comprises one of K, cys-Cys disulfide bonds. Wherein K is lysine and Cys is cysteine.
Preferably, the linker comprises one of Ahx, at least one glycine residue, polyethylene glycol. Wherein Ahx is aminocaproic acid.
The application also provides a preparation method of the denatured collagen targeted antibacterial peptide, which comprises the following steps: based on the sequence of the denatured collagen targeted antibacterial peptide, polypeptide solid-phase synthesis method is adopted for synthesis.
The method has the characteristics of convenient and quick synthesis, stability and controllability.
The application also provides application of the denatured collagen targeted antibacterial peptide or pharmaceutically acceptable salt thereof in preparation of antibacterial drugs.
Preferably, the bacterial species that the antibacterial agent inhibits/kills include escherichia coli, staphylococcus aureus, candida albicans, and pseudomonas aeruginosa.
The application also provides an antibacterial drug, which comprises the denatured collagen targeted antibacterial peptide or pharmaceutically acceptable salt thereof.
Preferably, the antibacterial drug further comprises pharmaceutically acceptable auxiliary materials.
Compared with the prior art, the application has the following beneficial effects:
according to the application, a plurality of antibacterial peptides (2 or more) are coupled with the collagen hybrid peptide in a parallel manner, so that an in-situ targeted wound antibacterial product is obtained, the antibacterial effect can be exerted on infected tissues in situ, efficiently and for a long time, the antibacterial effect is improved, and the side effect on normal tissues is reduced.
Drawings
FIG. 1 is a schematic diagram showing the principle of action of collagen hybrid peptides (Collagen hybridizing peptide, CHP); x: coupling drugs such as antibacterial peptides; CHP: collagen hybrid peptide.
FIG. 2 is a graph showing the results of an antimicrobial test for different types of conjugated peptides; wherein a-g respectively represent FLC, CHP, FLC-CHP, CHP-FLC3, (FLC) 2 ＝CHP、(FLC) 2 Antibacterial test results of CHP-FLC; -series, =parallel; the black bars in the bar graph represent staphylococcus aureus and the gray bars represent pseudomonas aeruginosa.
FIG. 3 is (FLC) 2 Antibacterial activity of CHP against staphylococcus aureus (Staphylococcus aureus), pseudomonas aeruginosa (Pseudomonas aeruginosa), escherichia coli (Escherichia coli) and Candida albicans (Candida albicans).
FIG. 4 is a graph showing the degree of affinity of different types of conjugated peptides for gelatin (denatured collagen) that mimics the structure of wound collagen; * *: p is less than 0.01.
FIG. 5 is a graph showing the targeting of different types of conjugated peptides to dermal layer unwinding collagen; white arrow: non-specific binding sites (hair follicles); yellow arrow: a epidermis; the rest positions are dermal tissues.
FIG. 6 is a graph showing the long-term efficacy of different types of conjugated peptides on targeting wound skin unwinding collagen; wherein 1-4 groups are wounded skin tissue; groups 5-6 are normal skin tissue; group 1 and group 5: (FLC) 2 =chp; group 2 and group 6: (FLC) 2 ＝ R CHP, wherein the sequence of CHP is disrupted; group 3: FLC; group 4: PBS.
Detailed Description
In order to make the technical solutions of the present application more apparent to those skilled in the art, the following examples will be presented. It should be noted that the following embodiments are only preferred embodiments of the present application, and the scope of the present application is not limited to the following embodiments, and any modifications, substitutions, and combinations made without departing from the spirit and principles of the present application are included in the scope of the present application.
The starting materials, reagents or apparatus used in the following examples are all available from conventional commercial sources or may be obtained by methods known in the art unless otherwise specified.
Rink Amide-AM resin (also, HCRAm 6-1-1); fmoc-Gly-OH (EMD Millipore, 852001); fmoc-flp-OH (Jier, GLS 200227-24175); fmoc-Hyp-OH (EMD Millipore, 852036); fmoc-6-Ahx-OH (An Naiji, A070322); fmoc-Lys (Boc) -OH (Jier, 71989-26-9); fmoc-L-Lys (Dde) -OH (Jil, 1069514); fmoc-Lys (Fmoc) -OH (Jil, GLS 210106-36810); fmoc-Phe-OH (Jier, GLS 210215-35701); fmoc-Leu-OH (Michelin, F809425); HATU (aladin, H109327); HOAt (Macklin, H811122); DIPEA (EMD Millipore, S1807494945); DMF (aladin, D112002); piperidine (de Tian Huagong, 20191105); 80% hydrazine hydrate (haze science, 200814); carboxyfluorescein (Sigma, 447293); DCM (Aladin, D116146); 1mL TFA (microphone, T818782); TIS (microphone, T819181); diethyl ether (guangzhou chemical reagent plant, 2020120510); acetonitrile (Macklin, a 800362); m9 liquid medium (Coolaber, SL 0060); SYTO9/PI live and dead bacteria staining kit (MKBio, MX 4234-40T); 4% paraformaldehyde (tetra and bio, SH 268C); goat serum (geto, ASL 038); DAPI (bi yun tian C1002); gelatin (Sigma, V900863); tween-20 (Soy Bao, T8220).
Example 1
This example provides a denatured collagen targeting antibacterial peptide (designated as (FLC) 2 =chp), its preparation method comprises the following steps:
(1) The polypeptide synthesizer is adopted to synthesize the gel by using Rink Amide-AM resin, glycine (G), fluoroproline (f) and hydroxyproline (O) as raw materialsPrimary hybrid peptide (GfO) 9 The sequence is as follows: gfOGFOGFOGFOGFOGFOGFOGFOGFOGFOGFO (SEQ ID NO: 1);
(2) At (GfO) 9 N-terminal amino caproic acid (Ahx) to Ahx- (GfO) 9 ；
(3) Starting from Fmoc-Gly-OH, fmoc-flp-OH and Fmoc-Hyp-OH, fmoc-6-Ahx-OH, fmoc-L-Lys (Dde) -OH, fmoc-Lys (Fmoc) -OH, fmoc-Phe-OH, fmoc-Leu-OH, fmoc-Lys (Fmoc) -OH and Fmoc-Lys (BOC) -OH in a polypeptide synthesizer in Ahx- (GfO) 9 The N-terminal of (C) is coupled with two antibacterial peptide FLC by using a connector, the two antibacterial peptide FLC are connected in parallel, and the Fmoc-Lys (Fmoc) -OH is used for K of the connection point to obtain (FLC) 2 ＝K-K(Dde)-Ahx-(GfO) 9 The method comprises the steps of carrying out a first treatment on the surface of the Wherein, 2 antibacterial peptides FLC are connected in parallel and then connected to two amino functional groups of the N end K; the sequence of the antibacterial peptide FLC is FLKLLKKLL (SEQ ID NO: 2);
( 4) With 5% hydrazine hydrate (80% hydrazine hydrate: dmf=6.25: 100 To (FLC) ) 2 ＝K-KDde-Ahx-(GfO) 9 Removing Dde protection of KDde; thereafter, the side chain amino-coupled fluorescein with Dde protected K removed from the resin can be selected for binding experiments.
(5) Washed with DMF (10 mL each, 3 replicates) and DCM (10 mL each, 3 replicates) and drained. With 1mL TFA/TIS/ddH 2 The mixture of O (volume ratio 95:2.5:2.5) is stirred for more than 3 hours at normal temperature. The TFA lysate was collected and the TFA was blown dry with nitrogen. Then adding 5mL of glacial ethyl ether to precipitate crude peptide, centrifuging (4 ℃ C., 4000rpm,4 minutes), removing supernatant, and repeating the precipitation washing steps twice to obtain a crude peptide sample;
(6) And purifying the polypeptide by reverse phase high performance liquid chromatography (Shimadzu, MALDI-8020). Chromatographic column (Agilent ZORBAX StableBond 300C 18), column incubator 85 ℃, mobile phase a (aqueous phase): ultrapure water with 0.1% tfa, mobile phase B (organic phase): acetonitrile containing 0.1% tfa. Linear gradient: 5-25 minutes: 5% -80% acetonitrile, procedure 30 min. The flow rate is: 4 mL/min, detection wavelength: 214nm and 254nm. Collecting the purified fraction, and freeze-drying (Freeze dryer, xinzhi, scientz-18N/C) to obtain (FLC) 2 ＝K-K-Ahx-(GfO) 9 (named (FLC) 2 ＝CHP)。
As can be seen from fig. 1, the principle of action of the denatured collagen-targeted antibacterial peptide is that the collagen hybrid peptide hybridizes with the triple helix of the unwound collagen molecule of a wound, an infectious inflammatory lesion, and anchors the coupled antibacterial peptide in the tissues of an infectious environment for long-acting drug efficacy.
Example 2: determination of antibacterial Activity
FLC, CHP, FLC-CHP (i.e., one antimicrobial peptide was coupled to the N-terminus of CHP), CHP-FLC (i.e., one antimicrobial peptide was coupled to the C-terminus of CHP), CHP-FLC3 (i.e., the C-terminus of CHP was coupled to 3 FLCs, and 3 FLCs were connected in series), were prepared by solid phase synthesis of polypeptides similar to example 1 2 =chp-FLC (i.e. at (FLC) 2 On the basis of =chp, 1 FLC was also attached to the C-terminal of CHP, and the same as in example 1 (FLC) 2 CHP together as antibacterial peptide material.
Bacterial culture: frozen strains (Staphylococcus aureus (Staphylococcus aureus), pseudomonas aeruginosa (Pseudomonas aeruginosa), escherichia coli (Escherichia coli) and Candida albicans (Candida albicans) were thawed and inoculated into centrifuge tubes of M9 basal medium, and cultured on a shaker (180 rpm) at 37℃for 16-18 hours.
Calculating the concentration of bacteria: the concentration of the bacterial solutions of staphylococcus aureus, pseudomonas aeruginosa, escherichia coli and candida albicans is adjusted to be 5 multiplied by 10 8 CFU/mL, OD at 600nm of about 0.08; diluting the bacterial liquid concentration to 1X 10 6 CFU/mL, ready for use.
80. Mu.L of the bacterial liquid and 20. Mu.L of the above-mentioned antibacterial peptide solution were added to each well of a 96-well plate, and the final concentrations of the antibacterial peptides were controlled to 1.56,3.13,6.25, 12.5, 25, 50, 100, 200. Mu.M, respectively. Media and blank groups (3 duplicate wells each) were set. After incubation in an incubator at 37℃for 16 hours, an enzyme-labeled instrument (BitoTek, synergy HTX) was used to measure OD at 600nm and to analyze the minimum inhibitory concentration (Minimal inhibitory concentration, MIC).
The test results are shown in fig. 2: the antibacterial peptide FLC has antibacterial property, and the minimum inhibitory concentration (MIC value) is 25 mu M; whereas CHP itself does not possess antibacterial properties; neither CHP-FLC nor FLC-CHP has antibacterial properties; CHP-FLC3 has relatively similar antimicrobial properties to FLC,the antibacterial performance is not effectively improved; (FLC) 2 Poor stability of CHP-FLC, easy precipitation in medium (see circled position on right panel in g), no antibacterial efficacy; only specific coupling products (FLC) 2 CHP gave a significant improvement in antimicrobial performance compared to FLC, with a MIC value of 12.5 μm. The above results indicate that not all coupling types produce better antibacterial properties, and that only coupling a plurality of antibacterial peptides (2 or more) in parallel with the collagen hybrid peptide can produce good effects.
Example 3: live and dead bacteria staining experiment
Evaluation by bacterial live-dead staining in example 1 (FLC) 2 Antibacterial activity of CHP against staphylococcus aureus, pseudomonas aeruginosa, escherichia coli and candida albicans. Mixing the bacterial liquid of the bacteria with (FLC) 2 Co-incubation for 1h with CHP, bacterial solution concentration 5×10 6 CFU/mL. After mixing SYTO9 and PI dyes (V SYTO9 :V PI =1:1), bacterial suspensions in the well plates were stained and incubated for 15 min at room temperature on a shaker protected from light. After centrifugation of the well plate (4000 rpm,1 min), imaging was performed with a fluorescence microscope (Thermo, EVOS M7000), imaging parameters: objective lens magnification: 20×; GFP (SYTO 9): light (0.02)/Ex (0.02 s)/Gain (20); RFP (PI): light (0.1)/Ex (0.2 s)/Gain (20).
The test results are shown in FIG. 3, (FLC) 2 CHP can kill staphylococcus aureus, pseudomonas aeruginosa, escherichia coli and candida albicans in a broad spectrum, and shows excellent antibacterial performance.
Example 4:
CHP (FLC) was prepared by solid-phase synthesis of polypeptides similar to example 1 2 =chp sum (FLC) 2 ＝ R CHP, wherein (FLC) 2 ＝ R CHP sequence (FLKLLKKLL) 2 =k-K-Ahx-offggofgfgfgfgfogofggooffg. And CHP (FLC) was labeled with Carboxyfluorescein (CF), respectively 2 =chp sum (FLC) 2 ＝ R CHP, the above polypeptides were used as test samples.
Preparing 10% (m/v) gelatin solution (1 XPBS buffer solution as solvent), heating at 70deg.C for 5 min to dissolve gelatin solution, sucking 100 μL gelatin solution, spreading on the bottom of 96-well plate with black transparent bottom, sucking rapidly, forming thin gelatin layer on the bottom of 96-well plate, and incubating at 4deg.C for 10 min to gel. 100. Mu.L of the above polypeptide sample was added per well at the concentration: 20. Mu.M, three duplicate wells were set and PBS was used as a blank control. After overnight incubation at 4℃each well was washed with 200. Mu.L of PBST (PBS containing 1% Tween-20 (v/v)) shaking (100 rpm,5 min) and repeated 3 times. The fluorescence intensity per well was measured with a microplate reader (Biotek, syne gy HTX), ex/Em:485/528nm, gain=35.
The test results are shown in FIG. 4, (FLC) 2 CHP exhibited higher than the disorder control peptide (i.e., FLC) 2 ＝ R CHP) and its affinity to the CHP itself, indicating (FLC) 2 CHP has the property of targeting denatured collagen.
Example 5: collagen targeting of SD rat skin tissue mass
Sample preparation: SD rat belly skin was obtained, tissue blocks (diameter 10 mm) were obtained by a skin biopsy punch, 4% paraformaldehyde solution was fixed overnight, and the tissue was divided into wound skin tissue and normal skin tissue, wherein the wound skin tissue was heated by water bath 100 ℃ for 10 minutes to make scalded wound.
Ex vivo tissue mass staining: with FLC, (FLC) 2 =chp sum (FLC) 2 ＝ R CHP was used as the test material (the polypeptides described above are as in example 4). Taking a 48-well plate, adding one isolated tissue block into each well, and adding 1mL of polypeptide solution (the concentration is 5 mu M); incubation at 4℃overnight, PBST (containing 1% Tween-20, 15 mL) was used to wash tissue blocks at 1,2,4,6,8, 10 hours, and 200. Mu.L of wash solution at various time points was collected and transferred to 96 well plates. Fluorescence value detection of the well plate was performed by a multimode microplate reader (Biotek, synergy HTX), ex/Em:485/528nm, gain=35.
Ex vivo tissue mass imaging: imaging of ex vivo skin tissue pieces by a small animal in vivo imager (PerkinElmer, IVIS lumine III) at different time points, imaging parameters: ex/Em:480/520nm; exposure:1s; fluorescent (Bing: 4;F/Stop: 2); photograph (Bing: 1;F/Stop: 8); field of view: C.
as shown in FIG. 5, after the skin of the rat is wounded, (FLC) 2 CHP can target the helicized collagen in the dermis of the skin, while FLC and (FLC) 2 ＝ R CHP has no targeting effect on the principle of dermal layer helicity of skin.
As shown in FIG. 6, after washing the rat skin tissue pieces with the buffer solution, fluorescence-labeled (FLC) 2 CHP can still remain in wounded skin tissue (shown in group 1 in fig. 6), indicating (FLC) 2 CHP can remain in tissue and continue to exert therapeutic effects.
The embodiments of the present application have been described in detail with reference to the accompanying drawings, but the present application is not limited to the above embodiments, and various changes can be made within the knowledge of one of ordinary skill in the art without departing from the spirit of the present application. Furthermore, embodiments of the application and features of the embodiments may be combined with each other without conflict.
Claims (5)
1. The denatured collagen targeting antibacterial peptide is characterized in that the coupling sequence from N end to C end in the denatured collagen targeting antibacterial peptide is (antibacterial peptide) x -branch point-linker-collagen hybrid peptide or collagen hybrid peptide-linker-branch point- (antibacterial peptide) x The method comprises the steps of carrying out a first treatment on the surface of the Wherein x is a positive integer not less than 2; and x antibacterial peptides are connected in parallel;
the antibacterial peptide is Feleucoin-K3, and the amino acid sequence of the antibacterial peptide is FLKLLKKLL; x=2;
the branching point is K, and K is lysine;
the connector is Ahx, and Ahx is aminocaproic acid;
the collagen hybrid peptide is (GfO) 9 The sequence is as follows: gfOGFOGFOGFOGFOGFOGFOGFOGFOGFOGFOGFOGFO the method comprises the steps of carrying out a first treatment on the surface of the;
wherein f is 4-fluoroproline, G is glycine, and O is hydroxyproline.
2. The method for preparing the denatured collagen targeted antibacterial peptide of claim 1, comprising the steps of: based on the sequence of the denatured collagen targeted antibacterial peptide, polypeptide solid-phase synthesis method is adopted for synthesis.
3. The use of a denatured collagen-targeted antibacterial peptide of claim 1 or a pharmaceutically acceptable salt thereof in the preparation of an antibacterial drug.
4. The use according to claim 3, wherein the bacterial species inhibited/killed by the antibacterial agent include escherichia coli, staphylococcus aureus, candida albicans and pseudomonas aeruginosa.
5. An antibacterial agent comprising the denatured collagen-targeted antibacterial peptide of claim 1 or a pharmaceutically acceptable salt thereof.
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