Multifunctional peptide design method and application thereof

Abstract

The invention discloses a multifunctional peptide design method and application thereof, the method comprises sequence allosteric operation, independent of splicing of different functional fragments, and capability of improving a single-function polypeptide to enable the single-function polypeptide to have multiple functions, and the specific method is as follows: on the tendency of a first functional template sequence, individual amino acid mutation is carried out to enable the first functional template sequence to be converted into second functional similarity; the polypeptide is endowed with multifunctionality and can be simplified and understood that a characteristic functional sequence and a characteristic polypeptide structure respectively perform different functions, so that the effect that a single sequence has multiple functions is achieved.

Description

Technical Field

[0001] The present invention relates to a novel multifunctional peptide design method, and specifically to a multifunctional peptide design method and application thereof. Background Art

[0002] For diseases with complex pathological characteristics and pathological processes, single-functional peptides can often only cancel one course of disease, one target, or one activity, and cannot adapt to the multiple needs of complex diseases. The multiple effects of multifunctional peptides act simultaneously, which can significantly improve the drugability and effectiveness of indications, maximize biological activity, and provide multiple solutions for key points of complex diseases, with good research and application prospects. Existing natural or synthetic multifunctional peptides have disadvantages such as functional incoordination, functional loss, and excessively high effective concentrations. Good design methods are needed to guide the design of multifunctional peptides.

[0003] Common multifunctional peptide design ideas include: 1) Linking: by introducing a linker, two active fragments are combined to perform the target function respectively; 2) Fusion: gradually reducing the size of the linker until the two functional fragments are directly coupled; 3) Chimerism: using the commonalities in the structure of the two target fragments to achieve merging.

[0004] However, compared with the activity of its template peptide, the multifunctional peptide often has significantly reduced functions. It can be seen that it is difficult to design a multifunctional peptide with good activity, that is, different functional polypeptide fragments interfere with each other due to charge, hydrophilicity, and structure, which leads to the complexity of the overall molecular structure, and requires continuous design and optimization to rationally transform the molecular structure.

[0005] Therefore, the inventor proposed an innovative design idea besides the traditional fragment splicing design idea. Summary of the invention

[0006] The technical problem to be solved by the present invention is to overcome the above technical defects and provide a method for designing a multifunctional peptide that does not require fragment splicing and can undergo structural transformation by replacing amino acids at individual sites, thereby enabling the polypeptide to acquire other functions.

[0007] In order to solve the above technical problems, the technical solution provided by the present invention is: a method for designing a multifunctional peptide, which adopts "sequence conformational change" for design, including mutating individual amino acids of a monofunctional template polypeptide to cause structural changes, and transforming the structure similarity to a second functional sequence with other functions, thereby obtaining a multifunctional peptide.

[0008] Preferably, the amino acid mutation of the monofunctional template polypeptide includes determining sequence characteristics similar to the second functional sequence, and performing amino acid mutation on the monofunctional template polypeptide after the similar sequence characteristics are determined.

[0009] Preferably, the multifunctional peptide is an osteogenic growth peptide, and the second functional sequence is an antimicrobial peptide; the osteogenic growth peptide is subjected to amino acid site replacement to obtain the multifunctional peptide, and the amino acid site replacement includes compliance with the hydrophilic-hydrophobic arrangement characteristics, positive charge and structural simplification principles.

[0010] Preferably, the amino acid site replacement includes replacing the QG of the amino acids at positions 5 and 6 of the OGP template with hydrophobic WL, replacing the amino acid G at position 11 with hydrophilic positive K, and replacing the amino acid A at position 1 with W or G, to obtain SEQ I and SEQ II, respectively.

[0011] Another aspect of the present invention discloses: application of a multifunctional peptide design method in a new design of multifunctional peptide research and development.

[0012] Another aspect of the present invention discloses: application of a multifunctional peptide design method in the research and development of new designs of osteogenic growth peptides.

[0013] Another aspect of the present invention discloses: application of SEQ I and SEQ II in antibacterial, immunomodulatory and osteogenesis.

[0014] The advantages of the present invention over the prior art are that, through sequence conformational modification, the present invention does not rely on the splicing of different functional fragments, and can improve a single-functional polypeptide to make it multifunctional, thereby achieving a single-sequence multifunctional effect, thereby solving the problems of existing multifunctional peptides, such as inconsistent functions, missing functions, and excessively high effective concentrations. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 It is a multifunctional peptide design method, peptide sequence and allosteric schematic diagram.

[0016] Figure 2 It is a structural diagram of the present invention when the patent name is used.

[0017] Figure 3 This is a schematic diagram of the osteogenic ability of new peptides using the multifunctional peptide design method. DETAILED DESCRIPTION

[0018] The present invention is further described in detail below in conjunction with the accompanying drawings.

[0019] The new method for designing multifunctional peptides of the present invention is shown as follows: Taking osteogenic growth peptide OGP as an example, the polypeptide has osteogenic activity and is a random coiled peptide. Through the new method for designing multifunctional peptides of "sequence conformational change", the polypeptide additionally obtains antibacterial ability and immunomodulatory ability, and obtains alpha helical multifunctional peptides SEQ I and SEQ II. Osteogenic growth peptide OGP has certain hydrophilic and hydrophobic alternating arrangement characteristics, which conforms to the amphiphilic tendency of certain antimicrobial peptides. The replacement and adjustment of hydrophilic and hydrophobic amino acids at individual sites make the overall sequence arrangement of osteogenic growth peptide close to the antimicrobial peptide, thereby obtaining antibacterial-immunomodulatory ability while retaining its osteogenic activity. For example, we found that OGP has hydrophilic and hydrophobic alternating arrangement characteristics, such as yellow shows that amino acids are hydrophobic, blue, pink and purple are hydrophilic, and the antimicrobial peptide sequence is positively charged (blue is a positively charged amino acid). Therefore, we found that although OGP has no antibacterial property, it has some antibacterial sequence characteristics. Relying on this characteristic, we replace individual amino acid sites in the sequence. According to the principle of structural simplification, the 5th and 6th amino acids were replaced by QG with hydrophobic WL in order to be more consistent with the hydrophilic and hydrophobic arrangement characteristics; the fourth-to-last G was replaced with a hydrophilic positively charged K in order to be more consistent with the hydrophilic and hydrophobic characteristics and enhance the positive charge; the 1st position A was replaced here considering that W and G often appear in the 1st position of antibacterial peptides. On the basis of utilizing and retaining the main sequence of OGP, we only made individual amino acid changes. The changed amino acids have a high homology with the parent peptide OGP sequence, but the structure and function of the polypeptide have changed significantly. This method, which does not rely on fragment splicing, utilizes the polypeptide's own tendencies and characteristics, simplifies the design process, and only makes a few amino acid replacements, can be used as a new and convenient multifunctional peptide design method. Replacement design methods, polypeptide sequences, and polypeptide conformational changes are as follows. Figure 1 As shown in Table 1, Figure 2 and Figure 3 This new and simple design and modification method gives OGP, a single-function osteogenic peptide, multiple functions of "antibacterial-immunomodulatory-osteopromoting". Compared with OGP, which can only be used for simple bone defects, the newly designed multifunctional peptide can be used for bone defects under pathological characteristics such as infection and inflammation. It has a wider range of applications, which also suggests the novelty and wide application of this design method.

[0020] 2. Antibacterial ability of multifunctional peptides:

[0021] The antibacterial ability of the multifunctional peptides designed by this novel method was evaluated by spot plate test using Gram-positive pathogenic bacteria Staphylococcus aureus and Gram-negative pathogenic bacteria Escherichia coli. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were detected by microplate dilution method drug sensitivity test.

[0022] ④Pick the preserved single colonies of Staphylococcus aureus and Escherichia coli and culture them aerobically in LB liquid culture medium at 37℃ constant temperature culture tank overnight.

[0023] ⑤ Pipette 100 μL of Staphylococcus aureus and Escherichia coli bacterial liquid into LB liquid medium and culture it aerobically in a constant temperature culture tank at 37℃. After subculturing for 2 generations, take the bacterial liquid in the middle logarithmic growth period and dilute it to 1×106 CFU / mL with MH medium for later use.

[0024] ⑥ The peptide was added to a 96-well plate using a 2-fold dilution method, 10 μL per well, and 90 μL of bacterial solution was added to make the final concentration of the peptide 2-128 μg / mL. Sterile water was used as a negative control.

[0025] ⑦The 96-well plate was placed in a 37°C constant temperature culture tank for aerobic culture for 24 hours.

[0026] ⑧MIC is the minimum inhibitory concentration of the clear wells in the plate.

[0027] ⑨Pipette 50 μL of bacterial solution from the clear well and spread it on the MH agar plate, and culture it aerobically overnight at 37°C in a constant temperature incubator for 24 hours. MBC is the minimum bactericidal concentration at which no colonies grow on the plate.

[0028] As shown in Table 1, OGP has no antibacterial ability, while SEQ I and SEQ II have good antibacterial ability against Staphylococcus aureus and Escherichia coli, proving that the multifunctional peptide design scheme endows the non-antibacterial peptide with antibacterial function.

[0029]

[0030] Table 1 Antibacterial ability of new peptides under the new multifunctional peptide design method

[0031] 3. Multifunctional peptide immunomodulatory ability:

[0032] The effects of the multifunctional peptides designed using this novel method on the expression of immune-inflammatory related genes iNOS, IL-1β, IL-6, Tnf-α, Arginase-1, and IL-4 in macrophages RAW246.7 were detected to evaluate their osteogenesis potential.

[0033] The specific steps are as follows:

[0034] (1) Macrophages were inoculated into 12-well plates and cultured in a CO2 incubator (5% CO2, 95% air, 100% humidity, 37°C) using high-glucose DMEM medium containing double antibodies and 10% fetal bovine serum.

[0035] (2) Macrophages were stimulated with 1 μg / mL E. coli LPS for 24 h to establish an inflammatory model, and the control group was incubated with a medium without LPS. Then the medium was replaced, and the LPS group was cultured with a medium containing LPS, the control group was cultured with a normal medium, and the peptide group was cultured with a medium containing peptide and LPS for another 24 h.

[0036] (3) Cells were collected for real-time quantitative PCR to measure the expression of immune-inflammatory related genes. RNA was isolated and purified using the RNA Rapid Extraction Kit (Yisan Biotechnology, Shanghai, China). RNA concentration and purity were measured using NanoDrop 2000 (Thermo Scientific, USA). cDNA was synthesized using the PrimeScriptTM Reverse Transcription Kit containing gDNA Eraser (TaKaRa, Japan). cDNA was used as a template qPCR Green Master Mix (Yisheng Biotechnology, Shanghai, China) Real-time quantitative PCR was performed using 480-II (Roche, Switzerland). β-actin was used as an internal reference, and the 2-ΔΔCt method was used to calculate the fold difference in gene expression.

[0037] like Figure 2 As shown, different lowercase letters indicate that there is a statistical difference in the expression of the gene in this group, P<0.05. The multifunctional peptide after this new design method has obtained significant immunomodulatory ability compared with the mother peptide OGP, and has a more obvious inhibitory effect on the pro-inflammatory related genes iNOS, IL-1β, IL-6, and Tnf-α, and a more obvious promoting effect on the anti-inflammatory related genes Arginase-1 and IL-4. Combined with Example 2, it can be seen that this simple "sequence allostery" new multifunctional peptide design method in the present invention not only gives the non-antibacterial mother peptide OGP antibacterial properties, but also gives it immunomodulatory function.

[0038] 4. Multifunctional peptides promote bone formation:

[0039] Alizarin red was used to detect the effect of multifunctional peptides on the osteogenic differentiation of bone marrow mesenchymal stem cells under this new design method.

[0040] The specific steps are as follows:

[0041] (1) BMSCs were seeded in 12-well plates and cultured in DMEM / F-12 medium containing double antibodies and 10% fetal bovine serum in a CO2 incubator (5% CO2, 95% air, 100% humidity, 37°C).

[0042] (2) The cultured adherent cells were cultured with fresh medium containing peptides every day. The negative control group used medium without antimicrobial peptides. The cells were cultured for 14 days.

[0043] (3) After 14 days, the bone marrow mesenchymal stem cells were fixed with glutaraldehyde and stained with Alizarin Red stain (Saiye, China), and the red mineralized nodules were used to characterize the osteogenic capacity.

[0044] like Figure 3As shown, SEQ I and SEQ II still retain obvious osteogenic ability compared to the parent peptide OGP, and there is no activity loss. Combined with Examples 2 and 3, the method imparts additional antibacterial and immunomodulatory activities to the parent peptide, suggesting that the novel multifunctional peptide design method of the present invention can impart other functions while retaining the original activity, and is a potential new method for designing multifunctional peptides.

[0045] Overcome: Complex diseases require multifunctional peptides to adapt to complex needs;

[0046] Existing multifunctional peptides often have drawbacks such as inconsistent functions, missing functions, and too high effective concentrations;

[0047] Existing multifunctional peptide design ideas often rely on fragment splicing, which can lead to design failures due to inconsistencies or conflicts in the physicochemical properties and activities between fragments.

[0048] The contents not described in detail in this specification belong to the prior art known to professional and technical personnel in this field.

[0049] The present invention and its embodiments are described above, and such description is not restrictive. The drawings show only one embodiment of the present invention, and the actual structure is not limited thereto. In short, if ordinary technicians in the field are inspired by it, without departing from the purpose of the invention, they can design a structure and embodiment similar to the technical solution without creativity, which should belong to the protection scope of the present invention.

Claims

1. A method for designing a multifunctional peptide, characterized in that: The design adopts "sequence conformational modification", which includes mutating individual amino acids in a single-functional template polypeptide to cause structural changes and transform the structural similarity to a second functional sequence with other functions, thereby obtaining a multifunctional peptide.

2. A multifunctional peptide design method according to claim 1, characterized in that: The amino acid mutation of the monofunctional template polypeptide includes determining the sequence characteristics similar to the second functional sequence, and performing amino acid mutation on the monofunctional template polypeptide after the similar sequence characteristics are determined.

3. A multifunctional peptide design method according to claim 1 or 2, characterized in that: The multifunctional peptide is an osteogenic growth peptide, and the second functional sequence is an antimicrobial peptide; the osteogenic growth peptide is subjected to amino acid site replacement to obtain the multifunctional peptide, and the amino acid site replacement includes compliance with the hydrophilic-hydrophobic arrangement characteristics, positive charge and structural simplification principles.

4. A multifunctional peptide design method according to claim 3, characterized in that: The amino acid site replacement includes replacing the QG of the amino acids at positions 5 and 6 of the OGP template with hydrophobic WL, replacing the amino acid G at position 11 with hydrophilic positive K, and replacing the amino acid A at position 1 with W or G, to obtain SEQ I and SEQ II, respectively.

5. Application of a multifunctional peptide design method in the new design of multifunctional peptide research and development.

6. Application of a multifunctional peptide design method in the development of new osteogenic growth peptide designs.

7. Use of SEQ I and SEQ II as described in claim 4 in antibacterial, immunomodulatory and osteogenesis.

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