Synthetic polypeptides and their use in the treatment of herpes virus infections
By designing peptides that specifically target the PRV gD receptor binding domain, the binding of the virus to the host cell receptor is blocked, solving the problems of immune escape and drug resistance of PRV and HSV in the prior art. This achieves a low-toxicity and high-efficiency antiviral effect and is suitable for the preparation of antiviral drugs.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- LANZHOU VETERINARY RESEARCH INSTITUTE CHINESE ACADEMY OF AGRICULTURAL SCIENCES(LANZHOU BRANCH CENTER OF CHINA ANIMAL HEALTH & EPIDEMIOLOGY CENTER)
- Filing Date
- 2026-04-21
- Publication Date
- 2026-08-04
AI Technical Summary
In existing technologies, PRV and HSV vaccines and traditional nucleic acid antiviral drugs have problems with immune escape and drug resistance, and nucleic acid drugs have liver and kidney toxicity. There is an urgent need to develop new inhibitors that are not dependent on viral enzymes and have low toxicity and high efficacy.
We designed and synthesized peptides gD_p1120, gD_p1036, and gD_p676 that specifically target the PRV gD receptor binding domain, blocking the binding of the virus to the host cell receptor Nectin-1, forming a high-affinity complex, and thus blocking viral infection.
The peptides have no significant cytotoxicity, possess highly efficient antiviral activity, and significantly inhibit the replication of PRV and HSV. They are suitable for the preparation of antiviral drugs and are widely used in animal husbandry and human treatment.
Smart Images
Figure CN122080134B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of biomedical technology, specifically to a synthetic polypeptide and its application in combating herpes virus infection. Background Technology
[0002] Pseudorabies virus (PRV) belongs to the alpha herpesvirus subfamily and can infect various mammals, including pigs, cattle, and sheep, with pigs being the primary host. Infection causes high fever and neurological symptoms in piglets and reproductive disorders in adult pigs. PRV mediates viral adsorption, invasion, and replication by binding to the host cell surface receptor Nectin-1 via the receptor-binding domain (RBD) of its envelope glycoprotein gD. Currently, PRV prevention and control mainly rely on vaccines and traditional nucleic acid antiviral drugs. However, PRV mutates rapidly, vaccines are prone to immune evasion, and nucleic acid antiviral drugs require viral thymidine kinase activation, which can induce drug-resistant mutants with long-term use and has hepatotoxic and nephrotoxic effects, thus limiting their clinical application.
[0003] Herpes simplex virus (HSV-1 / HSV-2) belongs to the same herpesvirus family as PRV. Its envelope glycoprotein gD is structurally highly similar to that of PRV gD, and its RBD domain is conserved. Both viruses rely on Nectin-1 as their primary host receptor, and their infection mechanisms are highly conserved. Existing anti-HSV drugs, such as acyclovir, require viral thymidine kinase activation, which easily leads to drug-resistant mutant strains.
[0004] In conclusion, there is an urgent need to develop a novel inhibitor that is not dependent on viral enzymes and is low in toxicity and highly effective. Summary of the Invention
[0005] To address the aforementioned shortcomings of existing technologies, the present invention aims to provide a synthetic polypeptide and its application in combating herpesvirus infection, thereby achieving highly efficient blocking of herpesvirus (PRV and HSV-1 / 2) infection.
[0006] The technical solution of the present invention to solve the above-mentioned technical problems is as follows: A synthetic polypeptide is provided, wherein the synthetic polypeptide is gD_p1120, gD_p1036, or gD_p676; wherein the amino acid sequence of gD_p676 is shown in SEQ ID NO.1; the amino acid sequence of gD_p1036 is shown in SEQ ID NO.2; and the amino acid sequence of gD_p1120 is shown in SEQ ID NO.3.
[0007] This invention provides an application of the above-mentioned synthetic polypeptide in the preparation of pharmaceutical formulations.
[0008] The present invention provides a pharmaceutical formulation comprising at least one of the above-mentioned gD_p1120, gD_p1036 and gD_p676.
[0009] Furthermore, pharmaceutical formulations also include pharmaceutically acceptable carriers or excipients.
[0010] Furthermore, the dosage form of the drug formulation is an injection, nasal spray, oral tablet, or sustained-release microsphere formulation.
[0011] This invention provides the application of the above-mentioned synthetic polypeptide or the above-mentioned pharmaceutical preparation in the preparation of antiviral drugs.
[0012] Furthermore, the virus is a herpesvirus.
[0013] Furthermore, herpesviruses include at least one of pseudorabies virus, herpes simplex virus type 1, and herpes simplex virus type 2.
[0014] The present invention has the following beneficial effects: (1) This invention designed and obtained three peptides, gD_p1120, gD_p1036, and gD_p676, that specifically target the key residues W135, L221, P223, F224, P229, and V233 of the PRV gD receptor binding domain (RBD). These three peptides can form high-affinity complexes with the PRV gD RBD, thereby specifically occupying the binding sites of the PRV gD RBD and Nectin-1.
[0015] (2) The three peptides of this invention showed no significant cytotoxicity. CCK8 assay results indicated that the half-maximal cytotoxicity concentration (MCC) of gD_p676 was [missing value]. 50 =2762.06μM; CC of gD_p1036 50 =3538.91μM; CC of gD_p1120 50 =5004.4 μM; the three peptides showed good biocompatibility and high anti-PRV activity. Flow cytometry quantitative analysis indicated that the half-maximal effective concentration (EC50) of gD_p676 was 5004.4 μM. 50 =77.83μM; EC of gD_p1036 50 =405.55μM; EC of gD_p1120 50 =255.08μM.
[0016] (3) Western blot analysis confirmed that all three peptides significantly inhibited PRV UL42 protein expression; meanwhile, TCID... 50 The results showed that all three peptides could significantly reduce PRV titers and effectively inhibit viral replication.
[0017] (4) The gD proteins based on HSV-1 / 2 are structurally highly similar to the PRV gD proteins, and their receptor-binding domains (RBDs) are conserved. The three peptides of this invention can also inhibit the replication of HSV-1 and HSV-2. Therefore, all three peptides of this invention can be used to prepare drugs against herpesvirus infection. Attached Figure Description
[0018] Figure 1 This is a schematic diagram showing three peptides targeting PRV gD RBD to block the binding of gD to Nectin-1; Figure 2 The graph shows the cytotoxicity results of the three peptides. Figure 3 The graph shows the inhibitory effects of three peptides on PRV-infected A549 cells. Figure 4 The graph shows the inhibition results of three peptides on PRV UL42 protein expression; Figure 5 The graph shows the inhibition results of three peptides on PRV titers. Detailed Implementation
[0019] The examples given below are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, conditions in the examples are performed under standard conditions or as recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.
[0020] Example 1: Synthesis and Cytotoxicity Assay of gD_p1120, gD_p1036 and gD_p676 (1) Synthesis: This invention utilizes the de novo peptide design process based on the RFdiffusion model to design and obtain three peptides specifically targeting the PRV gD receptor-binding domain (RBD): gD_p676 (DLELLRLYALS, SEQ ID NO.1), gD_p1036 (VSPWELEYLRRFEELLAEYLARAA, SEQ ID NO.2), and gD_p1120 (VDPYFVEYLERFLALLESYLAERA, SEQ ID NO.3). A schematic diagram illustrating the three peptides targeting the PRV gD RBD and blocking gD binding to Nectin-1 is shown below. Figure 1 Genscript Biotech Inc. was commissioned to synthesize three peptides. After HPLC purification, the purity of all three peptides was >95%, and the molecular weights identified by mass spectrometry were consistent with the theoretical values (the molecular weight of gD_p676 was 1305.54 Da, the molecular weight of gD_p1036 was 2925.34 Da, and the molecular weight of gD_p1120 was 2907.32 Da).
[0021] (2) Cytotoxicity assay: A549 cells were inoculated at 1×10⁻⁶ cells per cell line. 4 Cells were seeded at a density of 1 cell per well in 96-well plates. After cell adhesion, solutions of 0–10000 μM gD_p676, gD_p1036, and gD_p1120 were added to each well, and the plates were incubated at 37°C in a 5% CO2 incubator for 24 h. CCK8 reagent was then added to each well, and the plates were incubated for another 1 h. The absorbance (OD value) of each well was measured at 450 nm using a microplate reader to assess cytotoxicity.
[0022] Depend on Figure 2 It is evident that the three peptides of this invention showed no significant cytotoxicity within the experimentally determined concentration range, indicating that all three peptides possess good biosafety and can be used for subsequent antiviral experiments and potential clinical applications. The CCK8 assay results are as follows: the half-maximal cytotoxicity concentration (CMC) of gD_p676 is... 50 =2762.06μM; CC of gD_p1036 50 =3538.91μM; CC of gD_p1120 50 =5004.4μM.
[0023] Example 2: In vitro experiments on PRV infection using three peptides A549 cells were fed at a rate of 5 × 10⁻⁶ 4 Cells were seeded at a density of 1 cell / well in 48-well plates and cultured for 24 h until adherence. PRV-GFP (a recombinant fluorescent virus constructed from PRV strains via homologous recombination) was pre-incubated for 1 h with 0–1000 μM gD_p676, gD_p1036, and gD_p1120 solutions, respectively, and then seeded into each well (MOI=0.1). After 2 h of seeding, the virus solution was discarded and replaced with fresh culture medium. After culturing for another 24 h, PRV infection was observed, and cells and culture supernatant were collected for subsequent detection.
[0024] (1) Infection rate detected by flow cytometry Uninfected A549 cells were used as blank control (MOCK group). Cells from each group were collected, digested with trypsin, and GFP fluorescence intensity was detected using the B525-FITC channel of a Beckman flow cytometer. The infection rate of each group was recorded and analyzed, and the inhibition rate was calculated according to the formula: Inhibition rate = (infection rate of control group - infection rate of experimental group) / infection rate of control group × 100%.
[0025] Depend on Figure 3 It was found that all three peptides significantly inhibited PRV infection of A549 cells, and the inhibitory effect was concentration-dependent. Flow cytometry quantitative analysis showed the following: the half-maximal effective concentration (EC50) of gD_p676 was... 50=77.83μM, EC of gD_p1036 50 =405.55μM, EC of gD_p1120 50 =255.08 μM. The results showed that all three peptides had highly efficient anti-PRV activity.
[0026] 2. Western Blot detection of UL42 protein Cells were lysed and collected using 1×SDS cell lysis buffer, and whole-cell extracts were obtained by centrifugation. After quantification using a BCA protein quantification kit, SDS-PAGE electrophoresis was performed. After electrophoresis, proteins were transferred to nitrocellulose membranes, blocked with 5% skim milk, and then PRV UL42 primary antibody and GAPDH internal control primary antibody were added. The membranes were incubated overnight at 4°C. After washing with TBST buffer, HRP-labeled secondary antibody was added, and the membranes were incubated at room temperature for 1 hour. Finally, the protein bands were detected by chemiluminescence staining and gel imaging system.
[0027] The results are as follows Figure 4 As shown, compared with the control group, the brightness of the PRV UL42 protein band in the experimental group decreased in a dose-dependent manner, indicating that all three peptides could significantly reduce the expression level of PRV UL42 protein.
[0028] 3. TCID50 detection of PRV titer The PRV titer in cell culture supernatant was determined using the half-maximal tissue culture infection dose method (TCID50): the supernatant was serially diluted 10-fold with culture medium containing 2% serum and seeded into Vero cells (1×10⁶ cells / well) in 96-well plates. 4 (1 GFP / well); after inoculation, culture for 48-72 h, observe GFP-positive wells using a fluorescence microscope, and calculate PRV titer using the Reed-Muench method.
[0029] Depend on Figure 5 It can be seen that all three peptides can significantly reduce PRV titers, further confirming that they can effectively inhibit PRV replication.
[0030] The above results demonstrate that the three peptides designed in this invention can competitively bind to the PRV gD receptor-binding domain (RBD), blocking the interaction between viral gD and host cell surface Nectin-1, thereby efficiently blocking PRV infection and significantly inhibiting PRV replication. These peptides can be used to prepare drugs against PRV infection. Since the gD protein of herpes simplex virus is structurally highly similar to that of PRV gD protein, and their receptor-binding domains (RBDs) are highly conserved, the peptides of this invention can also inhibit the replication of herpes simplex virus type 1 (HSV-1) and herpes simplex virus type 2 (HSV-2), and can be used to prepare drugs against HSV infection.
[0031] In summary, the polypeptides of this invention are not only applicable to the prevention and treatment of PRV infection in animal husbandry, but also to the treatment of HSV infection in humans, and have broad market prospects and application value.
[0032] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.
Claims
1. A synthetic polypeptide, characterized in that, The artificially synthesized polypeptide is gD_p1120, gD_p1036, or gD_p676; wherein the amino acid sequence of gD_p676 is shown in SEQ ID NO.1; the amino acid sequence of gD_p1036 is shown in SEQ ID NO.2; and the amino acid sequence of gD_p1120 is shown in SEQ ID NO.
3.
2. The use of the synthetic polypeptide of claim 1 in the preparation of an anti-pseudorabies virus (PRV) infection agent.
3. An anti-pseudorabies virus (PRV) infection preparation, characterized in that, The formulation comprises at least one of gD_p1120, gD_p1036 and gD_p676 as described in claim 1.
4. The formulation according to claim 3, characterized in that, The formulation may also include pharmaceutically acceptable carriers or excipients.
5. The formulation according to claim 3 or 4, characterized in that, The dosage form of the preparation is an injection, nasal spray, oral tablet, or sustained-release microsphere formulation.
6. The use of the synthetic polypeptide of claim 1 or the formulation of any one of claims 3-5 in the preparation of a drug for treating pseudorabies virus (PRV) infection.