A polypeptide targeting formyl peptide receptor on cell membrane surface, a pharmaceutical composition and their applications

Abstract

The present application provides a polypeptide targeting formyl peptide receptor on the cell membrane surface, a pharmaceutical composition and their applications. The polypeptide targeting formyl peptide receptor on the cell membrane surface includes a ligand of the formyl peptide receptor and Cys or Lys located at the C-terminus of the polypeptide connected to the ligand of the formyl peptide receptor; or, the polypeptide is the ligand of the formyl peptide receptor, and the C-terminus of the ligand of the formyl peptide receptor is Cys or Lys. After the polypeptide stimulates the formyl peptide receptor on the cell membrane surface, the endocytosed formyl peptide receptor cannot be recycled to the cell membrane, thereby targeting and rapidly knocking down the formyl peptide receptor on the cell membrane surface. Moreover, by using this polypeptide to knock down the formyl peptide receptor on the cell membrane surface, there is no need to perform enzymatic modification on the polypeptide additionally, no need to rely on other degradation techniques, and the off-target effects of gene knockout and gene knockdown methods can be avoided. The operation is simple and the effectiveness is high.

Description

Technical Field

[0001] The present application relates to the field of biomedical technologies, and particularly to a polypeptide targeting formyl peptide receptor on the cell membrane surface, a pharmaceutical composition thereof, and their applications. Background Art

[0002] Formyl Peptide Receptor (FPR) is a member of the class A G-protein coupled receptor superfamily, and is divided into FPR1, FPR2, and FPR3. FPR is highly expressed on white blood cells, mainly distributed on the cell membrane, and is closely related to the occurrence and development of inflammation. FPR on the cell membrane can sense chemokines outside the cell. Bacterial infections, tissue damage, non-infectious inflammation, etc. can cause the release of chemokines, thereby mediating the migration of white blood cells to the infection site to kill bacteria. However, this effect has two sides. Moderate white blood cell migration is beneficial to the recovery of diseases, but excessive white blood cell migration will cause white blood cell infiltration and thus produce inflammation, which will be unfavorable for the recovery of diseases. Therefore, reducing or completely knocking out FPR on the cell membrane surface is beneficial to alleviating inflammation and related diseases, such as acute lung injury, colitis, rheumatoid arthritis, Alzheimer's disease, etc.

[0003] Currently, the commonly used methods for reducing or completely knocking out cell surface receptors are siRNA knockdown and gene knockout. However, these two methods have complex operation processes, are time-consuming, and have the risk of off-target effects. Their safety, stability, and selectivity need to be improved. Additionally, there is a method called Proteolysis targeting chimeras (PROTAC) for targeting the degradation of target proteins. The specific method is to connect the ligand of the target protein with an E3 ubiquitin ligase to form a hybrid small molecule with two functions. It can form a ternary complex of the target protein and the E3 ubiquitin ligase, and enable the target protein to enter the ubiquitination system for degradation. However, this method has a complex process. First, a suitable ligand needs to be found, and then the ligand and the E3 ubiquitin ligase need to be connected. Moreover, there is a risk that the binding ability of the ligand modified by the E3 ubiquitin ligase to the target protein will change. Summary of the Invention

[0004] The purpose of the present application is to provide a polypeptide targeting formyl peptide receptor on the cell membrane surface, aiming to solve the problems existing in the existing methods for knocking down formyl peptide receptor on the cell membrane surface.

[0005] To achieve the above purpose, the present application provides a polypeptide targeting formyl peptide receptor on the cell membrane surface, including the ligand of the formyl peptide receptor, and Cys or Lys located at the C-terminus of the polypeptide and connected to the ligand of the formyl peptide receptor;

[0006] Or, the polypeptide is the ligand of the formyl peptide receptor, and the C-terminus of the ligand of the formyl peptide receptor is Cys or Lys.

[0007] Preferably, the formyl peptide receptor includes: formyl peptide receptor 1 and / or formyl peptide receptor 2.

[0008] Preferably, the N-terminus of the ligand of the formyl peptide receptor includes Met with modification;

[0009] Preferably, the modification includes any one of formylation, acetylation, phosphorylation, glycosylation, succinylation, ubiquitination;

[0010] Preferably, the ligand of the formyl peptide receptor includes N-formyl-Met;

[0011] In an alternative embodiment, the ligand of the formyl peptide receptor includes N-formyl-Met-Leu-Phe, or N-formyl-Met-Ile-Phe-Leu, or N-formyl-Met-Ile-Val-Thr-Leu-Phe, or N-formyl-Met-Met-Tyr-Ala-Leu-Phe.

[0012] Preferably, there are 0 to 10 arbitrary amino acids connected between the ligand of the formyl peptide receptor and Cys or Lys at the C-terminus of the polypeptide;

[0013] Preferably, there are 0 to 5 arbitrary amino acids connected between the ligand of the formyl peptide receptor and Cys or Lys at the C-terminus of the polypeptide;

[0014] More preferably, there are 0 to 2 arbitrary amino acids connected between the ligand of the formyl peptide receptor and Cys or Lys at the C-terminus of the polypeptide.

[0015] Preferably, the polypeptide targeting the formyl peptide receptor on the cell membrane surface is selected from any one of the following amino acid sequences a to d:

[0016] a, N-formyl-Met-Leu-Phe-Cys;

[0017] b, N-formyl-Met-Leu-Phe-Ile-Cys;

[0018] c, N-formyl-Met-Leu-Phe-Ile-Ile-Cys;

[0019] d, N-formyl-Met-Ile-Phe-Leu-Cys.

[0020] In an alternative embodiment, the polypeptide is the ligand of the formyl peptide receptor, and the ligand of the formyl peptide receptor is N-formyl-Met-Leu-Phe-Ile-Ile-Lys.

[0021] The present application also provides a pharmaceutical composition of the polypeptide targeting the formyl peptide receptor on the cell membrane surface as described above.

[0022] Preferably, the pharmaceutical composition exerts a therapeutic effect by knocking down the formyl peptide receptor on the cell membrane surface; or,

[0023] the pharmaceutical composition exerts a therapeutic effect by inhibiting the migration of immune cells.

[0024] The present application also provides the use of the polypeptide targeting the formyl peptide receptor on the cell membrane surface as described above in the preparation of a drug for treating inflammation and inflammation-related diseases.

[0025] Preferably, the inflammation-related diseases include any one of acute lung injury, colitis, rheumatoid arthritis, and Alzheimer's disease.

[0026] Compared with the prior art, the beneficial effects of the present application include:

[0027] The polypeptide targeting the formyl peptide receptor on the cell membrane surface provided by the present application includes a ligand of the formyl peptide receptor and Cys or Lys located at the C-terminus of the polypeptide connected to the ligand of the formyl peptide receptor; or, the polypeptide is the ligand of the formyl peptide receptor, and the C-terminus of the ligand of the formyl peptide receptor is Cys or Lys. After the polypeptide stimulates the formyl peptide receptor on the cell membrane surface, the endocytosed formyl peptide receptor cannot be recycled to the cell membrane, thereby targeting and rapidly knocking down the formyl peptide receptor on the cell membrane surface. And by knocking down the formyl peptide receptor on the cell membrane surface with this polypeptide, there is no need to additionally perform enzymatic modification on the polypeptide, no other degradation techniques are required, and the off-target effects of gene knockout and gene knockdown methods can also be avoided. The operation is simple and the effectiveness is high. Description of the Drawings

[0028] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings required to be used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present application and should not be regarded as limiting the scope of the present application.

[0029] Figure 1 It is a graph of the experimental results of the binding of the polypeptide to FPR1;

[0030] Figures 2A to 2C It is a graph of the experimental results of the knockdown of FPR1 by the fMLFC polypeptide in HeLa-FPR1-GFP, RBL-FPR1, and dHL60 cells respectively;

[0031] Figure 2D It is a confocal laser microscope graph of the distribution of FPR1 in HeLa-FPR1-GFP cells;

[0032] Figure 3It is a diagram showing the experimental results of fMLFIC, fMLFIIC and fMLFIIK polypeptides on FPR1 knockdown;

[0033] Figure 4 It is a diagram showing the experimental results of the polypeptide on cell migration. Specific embodiments

[0034] As used herein, the terms:

[0035] "Prepared from" is synonymous with "comprising". The terms "comprising", "including", "having", "containing" or any other variation thereof as used herein are intended to cover non-exclusive inclusion. For example, a composition, step, method, article or device comprising the listed elements is not necessarily limited to those elements, but may include other elements not expressly listed or elements inherent to such composition, step, method, article or device.

[0036] When an equivalent, concentration, or other value or parameter is expressed as a range, a preferred range, or a range defined by a series of upper preferred values and lower preferred values, this should be understood to specifically disclose all ranges formed by any pairing of any range upper limit or preferred value with any range lower limit or preferred value, whether or not the range is separately disclosed. For example, when the range "1 to 5" is disclosed, the described range should be interpreted to include the ranges "1 to 4", "1 to 3", "1 to 2", "1 to 2 and 4 to 5", "1 to 3 and 5", etc. When a numerical range is described herein, unless otherwise indicated, the range is intended to include its end values and all integers and fractions within the range.

[0037] "And / or" is used to indicate that either or both of the stated circumstances may occur. For example, A and / or B includes (A and B) and (A or B).

[0038] This application provides a polypeptide targeting the formyl peptide receptor on the cell membrane surface, including a ligand of the formyl peptide receptor and a Cys or Lys located at the C-terminus of the polypeptide connected to the ligand of the formyl peptide receptor; or, the polypeptide is the ligand of the formyl peptide receptor, and the C-terminus of the ligand of the formyl peptide receptor is Cys or Lys.

[0039] The polypeptide targeting the formyl peptide receptor on the cell membrane surface provided by the present application includes a ligand of the formyl peptide receptor and a Cys or Lys located at the C-terminus of the polypeptide connected to the ligand of the formyl peptide receptor; or, the polypeptide is the ligand of the formyl peptide receptor, and the C-terminus of the ligand of the formyl peptide receptor is Cys or Lys. After the polypeptide stimulates the formyl peptide receptor on the cell membrane surface, the endocytosed formyl peptide receptor cannot be recycled to the cell membrane, thereby targeting and rapidly knocking down the formyl peptide receptor on the cell membrane surface. Moreover, by using this polypeptide to knock down the formyl peptide receptor on the cell membrane surface, there is no need to perform enzymatic modification on the polypeptide additionally, no other degradation techniques are required, and the off-target effects of gene knockout and gene knockdown methods can be avoided. The operation is simple and the effectiveness is high.

[0040] Preferably, the formyl peptide receptor includes: formyl peptide receptor 1 (FPR1) and / or formyl peptide receptor 2 (FPR2).

[0041] Preferably, the N-terminus of the ligand of the formyl peptide receptor includes a modified Met.

[0042] Preferably, the modification includes any one of formylation, acetylation, phosphorylation, glycosylation, succinylation, and ubiquitination.

[0043] Preferably, the ligand of the formyl peptide receptor includes N-formyl-Met.

[0044] In an alternative embodiment, the ligand of the formyl peptide receptor includes N-formyl-Met-Leu-Phe, or N-formyl-Met-Ile-Phe-Leu, or N-formyl-Met-Ile-Val-Thr-Leu-Phe, or N-formyl-Met-Met-Tyr-Ala-Leu-Phe.

[0045] Specifically, the ligand of the formyl peptide receptor including N-formyl-Met-Leu-Phe can be, for example, N-formyl-Met-Leu-Phe, N-formyl-Met-Leu-Phe-Lys, N-formyl-Met-Leu-Phe-Glu, N-formyl-Met-Leu-Phe-Trp, N-formyl-Met-Leu-Phe-Ile-Ile, N-formyl-Met-Leu-Phe-Ile-Ile-Lys, etc.

[0046] Preferably, 0 to 10 arbitrary amino acids are connected between the ligand of the formyl peptide receptor and the Cys or Lys at the C-terminus of the polypeptide;

[0047] Preferably, 0 to 5 arbitrary amino acids are connected between the ligand of the formyl peptide receptor and the Cys or Lys at the C-terminus of the polypeptide;

[0048] More preferably, there are 0 to 2 arbitrary amino acids connected between the ligand of the formyl peptide receptor and Cys or Lys at the C-terminus of the polypeptide.

[0049] Preferably, the polypeptide targeting the formyl peptide receptor on the cell membrane surface is selected from any one of the following amino acid sequences a to d:

[0050] a. N-formyl-Met-Leu-Phe-Cys;

[0051] b. N-formyl-Met-Leu-Phe-Ile-Cys;

[0052] c. N-formyl-Met-Leu-Phe-Ile-Ile-Cys;

[0053] d. N-formyl-Met-Ile-Phe-Leu-Cys.

[0054] In another alternative embodiment, the polypeptide is the ligand of the formyl peptide receptor, and the ligand of the formyl peptide receptor is N-formyl-Met-Leu-Phe-Ile-Ile-Lys. The applicant found that the ligand of the formyl peptide receptor, N-formyl-Met-Leu-Phe-Ile-Ile-Lys, can play a role in knocking down the formyl peptide receptor on the cell membrane surface. It can be understood that this solution does not exclude the polypeptide solution in which Cys or Lys is further connected to the C-terminus of N-formyl-Met-Leu-Phe-Ile-Ile-Lys.

[0055] The present application also provides a pharmaceutical composition of the above-mentioned polypeptide targeting the formyl peptide receptor on the cell membrane surface.

[0056] Preferably, the pharmaceutical composition exerts a therapeutic effect by knocking down the formyl peptide receptor on the cell membrane surface; or,

[0057] The pharmaceutical composition exerts a therapeutic effect by inhibiting the migration of immune cells.

[0058] The present application also provides the use of the above-mentioned polypeptide targeting the formyl peptide receptor on the cell membrane surface in the preparation of a drug for treating inflammation and inflammation-related diseases.

[0059] Preferably, the inflammation-related diseases include any one of acute lung injury, colitis, rheumatoid arthritis, and Alzheimer's disease.

[0060] The implementation scheme of the present application will be described in detail below in combination with specific embodiments. However, those skilled in the art will understand that the following embodiments are only used to illustrate the present application and should not be regarded as limiting the scope of the present application. For those not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. For reagents or instruments not specified for the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0061] Example 1 Polypeptide Preparation

[0062] The company synthesized the following polypeptides respectively: N-formyl-Met-Leu-Phe (fMLF, an endogenous ligand of FPR1), N-formyl-Met-Leu-Phe-Cys (fMLFC, fMLF plus C), N-formyl-Met-Leu-Phe-Ile-Cys (fMLFIC, fMLF plus IC), N-formyl-Met-Leu-Phe-Ile-Ile-Cys (fMLFIIC, fMLF plus IIC), N-formyl-Met-Ile-Phe-Leu-Cys (fMIFLC, endogenous ligand fMIFL plus C), N-formyl-Met-Leu-Phe-Ile-Ile-Lys (fMLFIIK, a ligand of FPR1).

[0063] Example 2 Polypeptide Binding Experiment with FPR1

[0064] Using the FPR1 endogenous ligand formyl tripeptide (N-formyl-Met-Leu-Phe, fMLF) as the control group and the fMLFC polypeptide prepared in Example 1 as the experimental group. The RBL-FPR1 stably transfected cells were suspended in HBSS buffer (containing 0.5% BSA and 20 mM Hepes) at a concentration of 1x10 5 cells / mL, placed on ice for 30 min, and then different concentrations of fMLFC polypeptide and 125 nM fMLFIIK-FITC mixture were added to the experimental group, and different concentrations of fMLF polypeptide and 125 nM fMLFIIK-FITC mixture were added to the control group. After mixing, they were placed on ice for 1 h, and then the mean fluorescence intensity of fMLFIIK-FITC in the cells of the experimental group and the control group was detected by flow cytometry respectively. fMLFIIK-FITC played a role in competing with the fMLFC polypeptide or fMLF polypeptide for binding to FPR1. The weaker the cell fluorescence intensity, the less fMLFIIK-FITC bound to the cells, the more the corresponding polypeptide bound, and the stronger the binding ability. The fluorescence intensity was converted into binding ability, and the results were as Figure 1 shown. It can be seen from Figure 1 that like the FPR1 endogenous ligand fMLF, the fMLFC polypeptide prepared in Example 1 can bind to FPR1, and its binding ability is stronger than that of fMLF.

[0065] Example 3: Experiment on the knockdown of FPR1 on the cell membrane surface by polypeptides

[0066] Using the endogenous ligand of FPR1, formyl tripeptide (N-Formyl-Met-Leu-Phe, fMLF), as the control group, fMLFC polypeptide as the experimental group, and the untreated group as the blank group. The experimental group and the control group polypeptides with a final concentration of 5 μM were used to stimulate the following three cell lines: HeLa-FPR1-GFP (transiently transfected with the FPR1-GFP plasmid to express FPR1-GFP in cells), RBL-FPR1 (a stable transfected cell line stably expressing FPR1), and dHL60 (HL60 cells were induced to become neutrophil-like cells after being induced with 1.3% DMSO for 5 days and express FPR1). Incubate at 37 °C for 1 h to induce FPR1 endocytosis, remove the polypeptides in the solution, and then incubate at 37 °C for 1 h to observe FPR1 recycled to the cell membrane. Use a fluorescent antibody against FPR1 to detect the number of FPR1 on the cell membrane by flow cytometry. The results of the three cell lines, HeLa-FPR1-GFP, RBL-FPR1, and dHL60, are respectively as Figures 2A to 2C shown, and it can be seen from Figure 2A Figure 2B and 2C that the fMLFC polypeptide can significantly reduce FPR1 on the cell membrane surface in the three cell lines. In addition, use HeLa-FPR1-GFP cells to observe the intracellular distribution of FPR1 under a laser confocal microscope. The results are as Figure 2D shown, and it can be seen from Figure 2D that in the blank group and the cells treated with fMLF, FPR1 is mainly distributed on the cell membrane, while after treatment with the fMLFC polypeptide, the distribution of FPR1 on the cell membrane is very little in the cells.

[0067] In addition, use fMLFIC, fMLFIIC, fMLFIIK, and fMIFLC polypeptides as the experimental groups respectively, and the untreated group as the control. The polypeptides with a final concentration of 5 μM were used to stimulate RBL-FPR1 cells. Incubate at 37 °C for 1 h to induce FPR1 endocytosis, remove the polypeptides in the solution, and then incubate at 37 °C for 1 h to observe FPR1 recycled to the cell membrane. Use a fluorescent antibody against FPR1 to detect the number of FPR1 on the cell membrane by flow cytometry. The results are as Figure 3 shown. According to Figure 3 Figure 3, it can be known that the fMLFIC, fMLFIIC, fMIFLC, and fMLFIIK polypeptides can also reduce the distribution number of FPR1 on the cell membrane in the cells.

[0068] Example 4: Experiment on the effect of polypeptides on cell migration

[0069] Using PBS buffer as the negative control and 10 nM fMLF as the positive control, after fMLF binds to FPR1, it will cause cell migration. The mixture of fMLFC polypeptide at different concentrations and 10 nM fMLF is the experimental group. Transfer dHL60 cells to the upper chamber of Transwell, and add PBS buffer, 10 nM fMLF, and the mixture of fMLFC polypeptide at different concentrations and 10 nM fMLF to the lower chamber culture medium respectively. After culturing at 37 °C for 2 hours, take the lower chamber culture medium and detect the number of cells migrated to the lower chamber with a flow cytometer. The results are as Figure 4 shown. It can be seen from Figure 4 this that fMLFC polypeptides at different concentrations can inhibit the migration of dHL60 cells.

[0070] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present application, rather than to limit them; although the present application has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present application.

[0071] In addition, those skilled in the art can understand that although some embodiments herein include certain features included in other embodiments but not others, the combination of features of different embodiments means that it is within the scope of the present application and forms different embodiments. For example, in the above claims, any one of the claimed embodiments can be used in any combination. The information disclosed in this background art section is only intended to deepen the understanding of the overall background art of the present application, and should not be regarded as an admission or any form of implication that this information constitutes prior art known to those skilled in the art.

Claims

1. A polypeptide targeting formyl peptide receptor on the cell membrane surface, characterized in that, The polypeptide targeting the formyl peptide receptor on the cell membrane surface is selected from any one of the following amino acid sequences b to d: b. N-formyl-Met-Leu-Phe-Ile-Cys; c. N-formyl-Met-Leu-Phe-Ile-Ile-Cys; d. N-formyl-Met-Ile-Phe-Leu-Cys.

2. A pharmaceutical composition comprising the polypeptide targeting the formyl peptide receptor on the cell membrane surface as described in claim 1.

3. Use of the polypeptide targeting the formyl peptide receptor on the cell membrane surface in the preparation of a drug for treating acute lung injury, wherein the polypeptide targeting the formyl peptide receptor on the cell membrane surface is selected from any one of the following amino acid sequences a to d: a. N-formyl-Met-Leu-Phe-Cys; b. N-formyl-Met-Leu-Phe-Ile-Cys; c. N-formyl-Met-Leu-Phe-Ile-Ile-Cys; d. N-formyl-Met-Ile-Phe-Leu-Cys.

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