CN107469067A - A kind of application of polypeptide and its variant in immunoregulation medicament is prepared - Google Patents

Abstract

The invention discloses the application of a polypeptide and its transformed body in the preparation of immunomodulatory drugs, belonging to the field of biotechnology. The amino acid sequence of the polypeptide is Gly-Nle-Arg-Pro-Tyr. The present invention modifies the short peptide and adds a permeability-enhancing part so that it can enter the interior of macrophages. For the first time, it is found that the polypeptide can specifically activate S1PR3 , significantly activate the MAPK pathway and enhance the bacterial clearance function of macrophages. Therefore, the polypeptide and its modified form can be applied to the preparation of immunomodulatory drugs, and provide transformation prospects for clinical infectious diseases targeting S1PR3 immunomodulatory therapy.

Description

Application of a polypeptide and its modified body in the preparation of immunomodulatory drugs

technical field

The invention relates to the field of biotechnology, in particular to the application of a polypeptide and its transformed body in the preparation of immunomodulatory drugs.

Background technique

G protein coupled receptor (GPCR) is a class of membrane receptors with seven transmembrane helices. It is the largest family of membrane receptor proteins in the human genome and is involved in almost all life activities in organisms. . This family is currently the most concerned target for clinical drug development, and about 1,000 proteins have become important targets for clinical drug treatment, accounting for about 50% of the market drugs. Among the current 50 best-selling marketed drugs, 20% belong to GPCR-related drugs.

GPCR proteins are abundantly expressed in body structural cells and immune cells, and play a key role in various pathophysiological processes, especially in anti-infection immunity. GPCR family members that have been reported to be involved in immune regulation include chemokine receptors (CXC chemokine receptors, CXCRs), 1-phosphorylated sphingosine receptors (Sphingosine 1-phosphate receptors, S1PRs), dopamine receptors and so on. It is an important way to solve the current antibiotic resistance by regulating the function of GPCR protein on the surface of immune cells and enhancing the body's own anti-infection ability.

As a major member of the S1PRs family, S1PR3 is coupled to three G proteins (Gi, Gq, G12/13) and mediates a wide range of cellular processes by activating mitogen-activated protein kinase (MAPK). Biological effects, such as regulating the production of cytokines and chemokines, promoting angiogenesis, etc. In particular, S1PR3 plays a key role in the regulation of acquired immunity and innate immunity: 1) In vivo studies have found that S1PR3 regulates the recruitment of B lymphocytes in the marginal zone of the spleen, and regulates the recruitment of MOMA1+ macrophages and MAdCAM-1+ endothelial cells in the marginal sinus. The linear arrangement of S1PR3 can form a natural barrier between the marginal zone and the germinal center, and the loss of S1PR3 can cause the decrease of B lymphocyte chemotaxis and the dysfunction of the spleen germinal center, leading to acquired immunodeficiency. In addition, S1PR3 promotes the recruitment of intestinal lamina propria dendritic cells to mesenteric lymph nodes, exerts antigen presentation effect, and mediates specific immunity. 2) In atherosclerosis, S1PR3 preferentially regulates the recruitment of anti-inflammatory monocytes to microvessels, enhances early tissue repair ability, and reduces the degree of atherosclerosis. Based on the monocyte HLA-DR% is a sensitive index reflecting the immune function of the patient, and its value is inversely proportional to the mortality rate. Our clinical study found that the expression level of S1PR3 in the monocyte HLA-DR high expression group of patients with sepsis was also higher. In addition, the expression level of monocyte S1PR3 was positively correlated with its bacterial clearance function. In summary, S1PR3 is an important molecule involved in immune regulation, and plays an important role in maintaining the body's immune balance, damage repair and anti-infection.

Currently, there is still a lack of specific agonists for S1PR3 on the market. Based on the fact that the second loop of GPCR cells plays an important role in signal transduction, it is a key path for clinical drug development to regulate receptor signal transduction by using peptides derived from the second loop of GPCR. Therefore, according to the amino acid sequence of the second intracellular loop of S1PR3, designing specific short peptides with high stability, strong solubility and transmembrane permeability will provide translational prospects for clinical infectious diseases targeting S1PR3 immune regulation therapy.

Contents of the invention

The purpose of the present invention is to provide a S1PR3-specific agonist short peptide with high stability, strong solubility and transmembrane permeability, so as to develop drugs for enhancing macrophage bacterial clearance and regulating immune function of the body.

To achieve the above object, the present invention adopts the following technical solutions:

The invention provides the application of the polypeptide whose amino acid sequence is Gly-Nle-Arg-Pro-Tyr in the preparation of immunomodulatory drugs.

The research of the present invention found for the first time that the transformation based on the sequence GY5 (Gly-Nle-Arg-Pro-Tyr) allows it to enter the interior of macrophages, specifically activates S1PR3, significantly activates the MAPK pathway, and significantly enhances macrophage bacterial clearance Function.

The present invention modifies and transforms the above-mentioned polypeptide in terms of stability, solubility, and transmembrane permeability, so as to improve its ability to act as a therapeutic drug.

The invention provides an application of a polypeptide transformation in the preparation of immunomodulatory drugs. The polypeptide transformation is obtained by transformation of a polypeptide whose amino acid sequence is Gly-Nle-Arg-Pro-Tyr.

The polypeptide engineer comprises a permeability enhancing moiety attached to the polypeptide via a direct bond or via a linker. Any moiety known in the art that actively or passively facilitates the entry of a compound into cells may be used for the modification of the permeability enhancing moiety, such as fatty acids, steroids, vitamins, carbohydrates, amino acids or transporter peptides. Preferably, the modified polypeptide comprises an arginine-rich polypeptide at its N-terminus. The arginine-rich polypeptide is linked to the amino terminus of the polypeptide directly or through a spacer.

The arginine-rich polypeptide is composed of 8-9 D-arginine residues. Preferably, the polypeptide is connected to the arginine-rich polypeptide by at least one glycine residue.

Through the above modification, the present invention obtains a modified polypeptide, the amino acid sequence of which is:

Arg ¹ -Arg ² -Arg ³ -Arg ⁴ -Arg ⁵ -Arg ⁶ -Arg ⁷ -Arg ⁸ -Arg ⁹ -Gly ¹⁰ -Gly ¹¹ -Nle ¹² -Arg ¹³ -Pro ¹⁴ -Tyr ¹⁵ .

Preferably, the C-terminal carboxyl group of the modified polypeptide is amidated to enhance the stability of the polypeptide chain, and it is named RY-15.

Another object of the present invention is to provide a pharmaceutical composition for immunoregulating macrophages to eliminate bacteria, including an effective amount of a polypeptide transformation and a pharmaceutically acceptable carrier, and the amino acid sequence of the polypeptide transformation is:

Arg ¹ -Arg ² -Arg ³ -Arg ⁴ -Arg ⁵ -Arg ⁶ -Arg ⁷ -Arg ⁸ -Arg ⁹ -Gly ¹⁰ -Gly ¹¹ -Nle ¹² -Arg ¹³ -Pro ¹⁴ -Tyr ¹⁵ .

The dosage of the modified polypeptide depends on the height, weight, age and health status of different patients. The effective dose refers to a sufficient dose to improve the symptoms of the treated object, and the improvement refers to reducing or alleviating the negative effects brought about by the diseased state during the treatment.

Active therapeutic ingredients are usually used together with pharmaceutical carriers, which can be reasonably selected according to the commonly used dosage forms of existing polypeptide drugs, such as sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, etc. Commonly used non-aqueous solvents include propylene glycol, polyethylene glycol, vegetable oils (such as olive oil), and organic esters for injection (such as ethyl oleate).

The beneficial effect that the present invention possesses:

In the present invention, the short peptide Gly-Nle-Arg-Pro-Tyr with the function of promoting angiogenesis is modified, and the permeability enhancement part is added so that it can enter the interior of macrophages. It is first found that the polypeptide can specifically activate S1PR3 and significantly activate MAPK pathway enhances bacterial clearance of macrophages. Therefore, the polypeptide and its modified form can be applied to the preparation of immunomodulatory drugs, and provide transformation prospects for clinical infectious diseases targeting S1PR3 immunomodulatory therapy.

Description of drawings

Figure 1 shows the distribution of FITC-RY15 detected by fluorescent confocal microscope after incubation of FITC-RY15 and THP-1 cells for 5min, 10min, 20min, and 30min respectively. Gradually increase.

Figure 2 shows the levels of total ERK and phosphorylated ERK (p-ERK) in THP-1 cells detected by immunoblotting respectively after treatment in solvent control group, GY5 group and RY15 group, as shown in the figure, compared with solvent control group and GY5 group , RY-15 can significantly increase the level of p-ERK in macrophages.

Figure 3 shows the bactericidal function of macrophages (BMDM) after the treatment of the solvent control group, GY5 group and RY15 group by the gentamicin protection test. As shown in the figure, compared with the solvent control group and the GY5 group, RY-15 can significantly increase Bactericidal ability of macrophages. *p<0.05.

detailed description

The present invention will be further described below in conjunction with specific examples.

Example 1

The amino acid sequence of GY5 is: Gly ¹ -Nle ² -Arg ³ -Pro ⁴ -Tyr ⁵ -NH ₂ .

GY5 is composed of 5 amino acids, and Nle is norleucine. The molecular weight of GY-5 is 846.3 Da.

The amino acid sequence of RY15 is:

Arg ¹ -Arg ² -Arg ³ -Arg ⁴ -Arg ⁵ -Arg ⁶ -Arg ⁷ -Arg ⁸ -Arg ⁹ -Gly ¹⁰ -Gly ¹¹ -Nle ¹² -Arg ¹³ -Pro ¹⁴ -Tyr ¹⁵ -NH ₂ .

RY15 is composed of 15 amino acids, and Arg is D-arginine. The molecular weight of RY15 is 2309.7 Da.

The amino acid sequence of FITC-RY15 is:

FITC-Arg ¹ -Arg ² -Arg ³ -Arg ⁴ -Arg ⁵ -Arg ⁶ -Arg ⁷ -Arg ⁸ -Arg ⁹ -Gly ¹⁰ -Gly ¹¹ -Nle ¹² -Arg ¹³ -Pro ¹⁴ -Tyr ¹⁵ -NH ₂

FITC is fluorescein isothiocyanate, which can emit green fluorescence under specific excitation light. Used for real-time imaging to detect the distribution of RY-15. Arg is D-arginine. The molecular weight of FITC-RY15 is 2699.0Da.

Example 2

The GY5, RY15 and FITC-RY15 of the above-mentioned Example 1 were synthesized by "Shanghai Jier Polypeptide Co., Ltd.". The success of the synthesis was verified by mass spectrometric analysis, and high performance liquid chromatography confirmed that the purity of both peptides was greater than 95%.

Example 3

Determination of the ability of the polypeptide RY15 to enter macrophages. The ability of peptides to enter macrophages was detected by fluorescent confocal microscopy.

The mononuclear macrophage cell line THP-1 was cultured with 1640 medium containing 10% fetal bovine serum, and the cells in the logarithmic phase were taken and added to a 24-well plate, with 5×10 ⁵ cells per well, and cultured in each well The base volume was 500 μl. The cells were incubated with FITC-RY15 at a final concentration of 10 μm for 5 minutes, 10 minutes, 20 minutes and 30 minutes, and the reaction was terminated with neutral formaldehyde. After the cells were resuspended, the cell suspension was spread on a glass slide, and after natural drying, DAPI was added to stain cell nuclei, and glycerol was added to mount the slides and observed and photographed under a fluorescent confocal microscope.

As shown in Figure 1, the green fluorescence intensity of the cytoplasm of THP-1 cells gradually increased as the time changed from 5 minutes to 30 minutes, confirming that the polypeptide RY15 entered into THP-1 cells in a time-dependent manner.

Example 4

Polypeptide RY15 can activate ERK, a key molecule in the MAPK pathway of macrophages, and increase its phosphorylation level.

The monocyte-macrophage cell line THP-1 was cultured with 1640 medium containing 10% fetal bovine serum, and the cells in logarithmic phase were taken, and the medium was replaced with pure 1640 medium. The cells were added to a 24-well plate, 5×10 ⁵ cells per well, and the volume of pure 1640 medium in each well was 500 μl. Peptides RY15 and GY5 were added respectively, with a final concentration of 10 μM. The blank control group was not added. After 10 minutes, the cells were aspirated into an EP tube, centrifuged at 4°C to remove the supernatant, and lysed with RIPA lysate. After the extracted protein was quantified by BCA method, the expression of p-ERK and ERK in each group was detected by immunoblotting.

As shown in Figure 2, the sequence GY5 obtained as the sequence modification of the intracellular functional region of S1PR3 cannot play the role of promoting ERK phosphorylation because it cannot enter the cell. The polypeptide RY15 relies on strong cell permeability and can significantly increase the level of ERK phosphorylation (p-ERK) in macrophages, thereby participating in the immune regulation function of the body.

Example 5

Polypeptide RY15 can promote the bactericidal function of macrophages

1) Take a C57 mouse and separate its tibia and femur. The bone marrow was washed out with PBS, the red blood cells were removed with red lysate, and cultured in DMEM medium containing 10% fetal bovine serum, 1% penicillin-streptomycin double antibody and 20ng/ml GM-CSF.

2) After overnight, take non-adherent cells and add them to a 6-well plate, and continue to culture for 7-8 days, wherein the same medium is used to change the medium once on the 4th day. The obtained cells are bone marrow derived macrophages (Bone Marrow Derived Macrophage, BMDM).

3) After the BMDM cells were digested and counted, 2×10 ⁵ cells per well were added to a 24-well plate, and each well contained 500 μl of culture medium. Allow cells to attach overnight.

4) The medium was replaced with pure DMEM, and after standing for 1 hour, 2×10 ⁶ Escherichia coli were added. Let stand in the cell culture incubator for 1 hour, so that the BMDM cells can phagocytize the E. coli.

5) Aspirate off the culture medium, wash it twice with PBS, add pure DMEM containing 100ng/ml gentamycin, 400 μl per well, and let it stand in the incubator for 1 hour, so that gentamicin can kill extracellular untreated cells. Phagocytosis of bacteria.

6) Add 10 μM of GY5 and RY15 respectively, add solvent to the control group, a total of 3 groups, and each group has 3 replicate wells. Put it in a bacterial incubator and let it stand for 6 hours.

7) Aspirate the supernatant, add PBS to wash twice, and then add PBS solution containing 0.5% Triton X-100 to lyse the cells and release the bacteria in the cells. After serially diluting, plate them, incubate overnight at 37°C, and count the colonies after they grow out.

As shown in Figure 3, the number of Escherichia coli in BMDM treated with RY15 was significantly less than that of the control group and GY5 treatment group, confirming that RY15 can promote macrophages to kill Escherichia coli.

sequence listing

<110> Zhejiang University

<120> Application of a Polypeptide and Its Modified Form in the Preparation of Immunomodulatory Drugs

<160> 2

<170> SIP Sequence Listing 1.0

<210> 1

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<221>MOD_RES

<222> (2)..(2)

<223> Nle

<220>

<221> UNSURE

<222> (2)..(2)

<223> The 'Xaa' at location 2 stands for Gln, Arg, Pro, or Leu.

<400> 1

Gly Xaa Arg Pro Tyr

1 5

<210> 2

<211> 15

<212> PRT

<213> Artificial Sequence

<220>

<221>MOD_RES

<222> (12)..(12)

<223> Nle

<220>

<221> UNSURE

<222> (12)..(12)

<223> The 'Xaa' at location 12 stands for Gln, Arg, Pro, or Leu.

<400> 2

Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Gly Gly Xaa Arg Pro Tyr

1 5 10 15

Claims (8)

1. amino acid sequence is application of the Gly-Nle-Arg-Pro-Tyr polypeptide in immunoregulation medicament is prepared.

2. a kind of application of polypeptide variant in immunoregulation medicament is prepared, the polypeptide variant are by amino acid sequence Gly-Nle-Arg-Pro-Tyr polypeptide transformation obtains.

3. application as claimed in claim 2, it is characterised in that the polypeptide variant also includes the rich smart ammonia positioned at its N-terminal Sour polypeptide.

4. application as claimed in claim 3, it is characterised in that described rich arginine polypeptide is by 8-9 D type arginine residues Composition.

5. application as claimed in claim 3, it is characterised in that by least one sweet between the polypeptide and rich arginine polypeptide Histidine residue is attached.

6. application as claimed in claim 5, it is characterised in that the amino acid sequence of the polypeptide variant is：

Arg¹-Arg²-Arg³-Arg⁴-Arg⁵-Arg⁶-Arg⁷-Arg⁸-Arg⁹-Gly¹⁰-Gly¹¹-Nle¹²-Arg¹³-Pro¹⁴- Tyr¹⁵。

7. application as claimed in claim 6, it is characterised in that the C-terminal carboxy amidation of the polypeptide variant.

8. a kind of pharmaceutical composition of immunological regulation macrophage bacteria removal, it is characterised in that the polypeptide including effective dose changes Body and pharmaceutically acceptable carrier are made, the amino acid sequence of the polypeptide variant is：

Arg¹-Arg²-Arg³-Arg⁴-Arg⁵-Arg⁶-Arg⁷-Arg⁸-Arg⁹-Gly¹⁰-Gly¹¹-Nle¹²-Arg¹³-Pro¹⁴- Tyr¹⁵。