Synthetic polypeptide p2 and its use in the treatment of herpes virus infections

Abstract

The application belongs to the field of biological medicine, and particularly relates to a synthetic polypeptide P2 and application thereof in anti-herpes virus infection. The polypeptide P2 which is specific to key amino acid residues (77N, 80I, 82N, 84N, 85N, 129F) of a virus glycoprotein gD combined with a host cell surface receptor Nectin-1 is obtained by an RFdiffusion algorithm design; the polypeptide P2 can significantly inhibit PRV infection and has no significant cytotoxicity; the polypeptide P2 can significantly down-regulate PRV UL42 protein expression and gB mRNA level; based on the conservation of Nectin-1 in the herpes virus invasion mechanism, the polypeptide P2 can also be used for inhibiting infection of other herpes viruses (for example, HSV-1 and HSV-2), and provides an important strategy for developing a new anti-herpes virus drug.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of biological medicine, and particularly relates to an artificially synthesized polypeptide P2 and application thereof in anti-herpes virus infection. BACKGROUND

[0002] Pseudorabies virus (PRV) belongs to the alpha herpes virus subfamily, and can infect pigs, cattle and other mammals, causing nervous system damage and significant economic losses. PRV invades through the binding of its envelope glycoprotein gD to the host cell surface receptor Nectin-1. At present, PRV prevention and control mainly relies on vaccines and nucleic acid inhibitors, but there are problems such as immune escape, drug resistance and liver and kidney toxicity.

[0003] Herpes simplex virus (HSV-1 / HSV-2) also relies on Nectin-1 as the main host receptor, and its infection mechanism is highly conserved with PRV. Existing anti-HSV drugs such as acyclovir need to rely on viral thymidine kinase activation, which is easy to cause drug-resistant mutant strains, and new inhibitors that do not depend on viral enzymes are urgently needed.

[0004] Studies have shown that the gD binding domain of Nectin-1 key residues (77N, 80I, 82N, 84N, 85N, 129F) are the molecular "hot spots" of viral invasion. Based on this, the application designs a polypeptide P2, which precisely binds to the above-mentioned sites through spatial conformation optimization, and blocks virus adsorption and internalization. SUMMARY

[0005] Based on the above technical problems, the application provides an artificially synthesized polypeptide P2, which competes with the interaction of viral gD and host Nectin-1, and efficiently blocks herpes virus (PRV and HSV) infection, and specifically includes the following contents:

[0006] In a first aspect, the application provides an artificially synthesized polypeptide P2, and the amino acid sequence of the polypeptide P2 is: PSVPSTSPISELSKLLEELLEELLG.

[0007] In a second aspect, the application provides the use of the polypeptide P2 in the preparation of an antiviral drug.

[0008] Preferably, the virus is a herpes virus.

[0009] Preferably, the herpes virus is selected from the group consisting of pseudorabies virus, herpes simplex virus type 1, and herpes simplex virus type 2.

[0010] Preferably, the herpes virus is pseudorabies virus.

[0011] Preferably, the polypeptide P2 inhibits virus adsorption to host cells or blocks viral genome replication.

[0012] Preferably, the polypeptide P2 blocks the interaction of viral glycoprotein gD with Nectin-1 to inhibit viral infection.

[0013] Preferably, the polypeptide P2 specifically targets the key residues 77N, 80I, 82N, 84N, 85N, 129F of the gD binding domain of the host cell surface receptor Nectin-1.

[0014] In a third aspect, the present application provides a pharmaceutical composition, wherein the active ingredient of the pharmaceutical composition comprises the polypeptide P2 according to the first aspect.

[0015] In a fourth aspect, the present application provides use of the pharmaceutical composition according to the third aspect in the preparation of an antiviral drug.

[0016] Preferably, the virus is a herpes virus.

[0017] Preferably, the herpes virus is selected from the group consisting of pseudorabies virus, herpes simplex virus type 1, and herpes simplex virus type 2.

[0018] Preferably, the herpes virus is pseudorabies virus.

[0019] In a fifth aspect, the present application provides a pharmaceutical preparation, wherein the pharmaceutical preparation comprises the polypeptide P2 according to the first aspect or the pharmaceutical composition according to the third aspect and a pharmaceutically acceptable carrier or excipient.

[0020] Preferably, the dosage form of the pharmaceutical preparation comprises an injection, a nasal spray, an oral tablet, or a sustained-release microsphere preparation.

[0021] In a sixth aspect, the present application provides use of the pharmaceutical preparation according to the fifth aspect in the preparation of an antiviral drug.

[0022] Preferably, the virus is a herpes virus.

[0023] Preferably, the herpes virus is selected from the group consisting of pseudorabies virus, herpes simplex virus type 1, and herpes simplex virus type 2.

[0024] Preferably, the herpes virus is pseudorabies virus.

[0025] The beneficial effects of the present application are:

[0026] (1) The polypeptide P2 of the present application is obtained by RFdiffusion algorithm design, and specifically targets the key amino acid residues (77N, 80I, 82N, 84N, 85N, 129F) in the virus glycoprotein gD binding to the host cell surface receptor Nectin-1; the polypeptide P2 forms a high-affinity complex with the gD binding domain of Nectin-1 (predicted binding free energy ΔG = -8.6 kcal / mol);

[0027] (2) The polypeptide P2 of the present application significantly inhibits PRV infection of Hela and A549 cells at a concentration of 400 μM, with inhibition rates of 66% and 67%, respectively, and no cytotoxicity (CCK8 assay);

[0028] (3) The polypeptide P2 can reduce the expression of PRV UL42 protein and the level of gB mRNA, indicating that it simultaneously inhibits the early adsorption and late replication stages of the virus.

[0029] (4) Based on structural similarity, the polypeptide P2 of the present application can also inhibit the replication of HSV-1 and HSV-2, and can be used for preparing a drug for treating herpes virus infection. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 Schematic diagram of polypeptide P2 targeting the gD binding domain of Nectin-1;

[0031] Figure 2 Results of CCK8 method for detecting cytotoxicity of polypeptide P2 (concentration 0-400 μM);

[0032] Figure 3 Results of fluorescence microscope observation of PRV infected cells (green fluorescently labeled virus);

[0033] Figure 4 Results of flow cytometry quantitative PRV infection inhibition rate;

[0034] Figure 5 Results of Western blot detection of PRV UL42 protein expression;

[0035] Figure 6 Results of qPCR detection of PRV gB mRNA level. DETAILED DESCRIPTION

[0036] The experimental methods in the following examples are all conventional methods unless otherwise specified; the experimental materials used in the following examples are all obtained from conventional biochemical reagent companies unless otherwise specified.

[0037] Example 1 Synthesis of polypeptide P2 and cytotoxicity determination

[0038] 1. Synthesis of polypeptide P2

[0039] In this example, polypeptide P2 (PSVPSTSPISELSKLLEELLEELLG) was designed by RFdiffusion algorithm to specifically target the key amino acid residues (77N, 80I, 82N, 84N, 85N, 129F) in the binding of viral glycoprotein gD to host cell surface receptor Nectin-1. The schematic diagram of polypeptide P2 targeting the gD binding domain of Nectin-1 is shown in Figure 1 .

[0040] The artificial synthesis of polypeptide P2 was commissioned to Kings River Biotech Co., Ltd. After HPLC purification, the purity of polypeptide P2 was >97%, and the mass spectrometry identified the molecular weight consistent with the theoretical value (2681.08 Da).

[0041] 2. Cytotoxicity

[0042] Hela cells were seeded in 96-well plates (1 x 10 4 / well), and 0-400 μM polypeptide P2 (containing 0.24% DMSO) was added, respectively. After incubation for 24 h, CCK8 reagent was added, and the absorbance was detected at 450 nm.

[0043] The results are shown in Figure 2 , polypeptide P2 had no significant effect on the activity of Hela cells, and had good safety.

[0044] Example 2 Inhibition of PRV infection by polypeptide P2 in vitro

[0045] Hela and A549 cells were seeded in 48-well plates at a density of 5 x 104 / well, and cultured for 24 h. Then, 400 μM polypeptide P2 was pre-incubated for 2 h, followed by inoculation of PRV-GFP (MOI = 0.1). PRV-GFP was derived from a recombinant virus constructed using homologous recombination technology based on a PRV strain isolated in the laboratory. After 1 h, fresh medium was replaced, and 24 h later, PRV infection was observed and cells were collected.

[0046] 1. Fluorescence microscope observation of PRV infection

[0047] The cell culture plate inoculated with PRV-GFP for 24 h was placed on the fluorescence microscope stage, and the cell morphology and PRV-GFP infection were observed under ordinary light and fluorescence, respectively.

[0048] The results are shown in Figure 3 , polypeptide P2 could significantly reduce the number of PRV-infected cells (green fluorescently labeled virus), and significantly reduce PRV infection.

[0049] 2. Flow cytometry detection of infection rate

[0050] The fluorescence intensity of GFP was detected by B525-FITC channel of Beckman flow cytometer, and the infection rate of each group of cells (reflected by the proportion of GFP positive cells) was recorded and analyzed.

[0051] As shown in Table 1, polypeptide P2 can significantly inhibit PRV infection of Hela and A549 cells, and the inhibition rates are 66% and 67%, respectively. Figure 4

[0052] 3. Western blot detection of UL42 protein

[0053] The cells were lysed by cell lysis solution (1x SDS buffer), and the whole cell extract was quantified and subjected to SDS-PAGE analysis, and the results were transferred to a nitrocellulose membrane. The primary antibodies of UL42 and GAPDH were used for blotting, and the HRP-labeled secondary antibody was used for chemiluminescence detection.

[0054] As shown in Table 2, polypeptide P2 can significantly reduce the expression of UL42 protein of PRV, and inhibit the early adsorption of PRV. Figure 5

[0055] 4. qPCR detection of gB mRNA

[0056] Total RNA was extracted from cells using TRIzol reagent, and cDNA was synthesized by Hiscript II qPT SuperMix II. Real-time fluorescent quantitative PCR analysis was carried out by QuantStudio 5 and SYBR RT-PCR kit, and the relative expression of RNA was calculated according to 2 -ΔΔCt The calculation method normalizes the relative expression of RNA to GAPDH.

[0057] As shown in Table 3, polypeptide P2 can significantly reduce the gB mRNA level of PRV, and the inhibition rates of PRV infection of Hela and A549 cells are 70% and 88%, respectively, which significantly inhibit the late replication of PRV. Figure 6 The above experimental results show that the polypeptide P2 described in the present application can effectively block PRV infection by competitively inhibiting the interaction between viral gD and host Nectin-1, significantly inhibit the replication of PRV, and can be used for preparing a drug against PRV infection.

[0058]

[0059] ​​​Since Herpes simplex virus (HSV-1 / HSV-2) also depends on Nectin-1 as the main host receptor, its infection mechanism is highly conserved with PRV, and therefore the polypeptide P2 described in the application can also inhibit HSV replication and can be used for preparing a medicine for resisting HSV infection. Therefore, the polypeptide P2 described in the application is suitable for the treatment of livestock breeding and human herpes virus infection, and has a broad market prospect.

Claims

1. A synthetic polypeptide P2, characterized in that, The amino acid sequence of the polypeptide P2 is: PSVPSTSPISELSKLLEELLEELLG.

2. A pharmaceutical composition, characterized by, The active ingredient of the pharmaceutical composition includes the polypeptide P2 as described in claim 1.

3. A pharmaceutical preparation, characterized by, The pharmaceutical preparation comprises the polypeptide P2 of claim 1 or the pharmaceutical composition of claim 2 and a pharmaceutically acceptable carrier or excipient.

4. The pharmaceutical preparation according to claim 3, characterized in that The dosage forms of the pharmaceutical preparations include injections, nasal sprays, oral tablets, or sustained-release microspheres.

5. The use of the polypeptide P2 of claim 1, the pharmaceutical composition of claim 2, or the pharmaceutical formulation of claim 3 in the preparation of an anti-pseudorabies virus drug.

6. Use according to claim 5, wherein The polypeptide P2 inhibits viral particle adsorption to host cells or blocks viral genome replication.

7. The application as described in claim 5, characterized in that, The polypeptide P2 blocks the interaction between viral glycoprotein gD and Nectin-1, inhibiting viral infection.

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