Application of RAD51 inhibitor in preparation of anti-pseudorabies virus drug

Abstract

The invention discloses an application of an RAD51 inhibitor in preparation of an anti-pseudorabies virus drug, belongs to the technical field of biological medicines, and particularly discloses a novel application of a small molecule compound RAD51 inhibitor B02 and DIDS in preparation of the anti-pseudorabies virus drug. The small molecule compounds provided by the scheme have anti-pseudorabies virus activity, the small molecule compounds can reduce the replication of PRV in A549 cells, and meanwhile, one of the small molecule compounds B02 can also significantly inhibit the replication of PRV in PK-15 cells; the compound can provide a theoretical basis and an innovative direction for screening and developing anti-PRV (pseudorabies virus) drugs, and has important scientific significance and potential application value in the field of research and development of anti-PRV drugs.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to the use of a RAD51 inhibitor in the preparation of an anti-pseudorabies virus drug. Background Art

[0002] Pseudorabies virus (PRV) belongs to the family Herpesviridae, subfamily Alphaherpesvirinae, genus Varicellavirus. PRV shares morphological characteristics with other herpesviruses, with virions approximately 110-225 nm in diameter and a round or oval shape with icosahedral symmetry. The virus is composed of four distinct structural structures: a linear double-stranded DNA genome, an icosahedral protein capsid, a protein envelope, and a lipid envelope containing viral glycoproteins. To date, multiple glycoproteins have been identified, most of which are components of the viral envelope. PRV spreads rapidly within pig farms, causing widespread infection and an acute epidemic. However, clinical symptoms of PRV infection vary among pigs of different ages and sexes. Infection in sows can lead to reproductive impairments, such as miscarriage, stillbirth, or mummification; semen quality in breeding boars can decline, leading to infertility; and respiratory problems and stagnant weight gain in fattening pigs. In piglets under two weeks of age, infection can result in a 100% mortality rate. Pseudorabies virus can also infect a variety of livestock, including cattle, sheep, cats, rabbits, and dogs, as well as wild animals such as minks and foxes. Notably, approximately 20 cases of human infection with pseudorabies virus have been reported in recent years, accompanied by intraocular inflammation, fever, seizures, encephalitis, and other severe central nervous system symptoms.

[0003] Maintaining DNA integrity is crucial for cellular function. However, DNA damage is commonly caused by exogenous factors (i.e., radiation, ultraviolet light, and chemical reagents) and endogenous factors (i.e., reactive oxygen species, lipid peroxidation, endogenous estrogens, and alkylating agents). DNA damage typically manifests as single-strand breaks (SSBs) or double-strand breaks (DSBs) and requires repair before DNA replication can initiate. Among all types of DNA damage, double-strand breaks are the most detrimental, and their repair primarily involves non-homologous end joining (NHEJ) and homologous recombination (HR). RAD51 is a central molecule in HR, involved in signaling pathways such as the DNA damage response and the cell cycle. The RAD51 gene is located on chromosome 15 and consists of nine exons and eight introns, encoding a 339-amino acid polypeptide. The DNA molecule is approximately 30 kb in size and exhibits DNA-dependent ATP-activated activity. It is one of the key enzymes involved in DNA homologous recombination and plays a crucial role in maintaining genomic stability. RAD51 is a key protein in the DNA double-strand damage repair pathway, playing an important role in repairing double-strand damage and maintaining genomic stability. Interestingly, RAD51 is also implicated in the transmission of viruses, including human immunodeficiency virus (HIV), human papillomavirus (HPV), hepatitis B virus (HBV), and hepatitis C virus (HCV).

[0004] The virulence and immunogenicity of PRV variants vary compared to traditional PRV, resulting in some commercial live vaccines being unable to safely protect susceptible pig populations. Furthermore, the efficacy of existing vaccines in humans and other susceptible animals remains unclear, and there is a lack of human anti-PRV drugs. This has led to the occurrence and spread of pseudorabies, a serious threat to pig production and public health safety. The development of effective drugs specifically targeting PRV is urgently needed. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to propose the use of RAD51 inhibitors in the preparation of anti-pseudorabies virus drugs, which can overcome the defects and shortcomings of traditional pseudorabies virus vaccines that lack protective efficacy, and use a class of small molecule compounds to prepare anti-pseudorabies virus infection drugs. The provided small molecule compounds have anti-pseudorabies virus activity.

[0006] In order to achieve the above technical objectives, the technical solution adopted by the present invention is:

[0007] Application of RAD51 inhibitors in the preparation of anti-pseudorabies virus drugs.

[0008] Wherein, the RAD51 inhibitor is the RAD51 inhibitor DIDS and / or the RAD51 inhibitor B02.

[0009] In addition, this proposal also proposes the use of RAD51 inhibitors in the preparation of drugs that inhibit the replication of pseudorabies virus in cells.

[0010] Wherein, the RAD51 inhibitor is the RAD51 inhibitor DIDS and / or the RAD51 inhibitor B02.

[0011] In addition, the cells are A549 cells and / or PK-15 cells

[0012] By adopting the above technical solution, the present invention has the following beneficial effects compared with the prior art:

[0013] This protocol uses CCK8 experiments to demonstrate that the RAD51 inhibitor DIDS has no toxic effect on cells at concentrations of 1-40 μM. The RAD51 inhibitor B02 also has no toxic effect on cells at concentrations of 1-20 μM, but has a certain toxic effect on cells at a concentration of 40 μM.

[0014] In addition, RT-PCR and RT-qPCR results showed that the RAD51 inhibitor B02 and DIDS groups significantly reduced the transcriptome levels of PRV genes in A549 cells compared to the DMSO group. RT-PCR further validated the inhibitor's function in PK-15 cells, demonstrating that the RAD51 inhibitor B02 could inhibit the expression of PRV genes gE and gD at the mRNA level.

[0015] In summary, this scheme can illustrate the function of RAD51 inhibitors DIDS and B02 in inhibiting pseudorabies virus replication and provide new drug options for the prevention and treatment of pseudorabies virus. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0017] Figure 1DIDS (a RAD51 inhibitor Figure 1 A), B02( Figure 1 B) Statistical graph showing different degrees of cytotoxicity in A549 cells; ns indicates no significant difference (P>0.05), and * indicates significant difference (P<0.05).

[0018] Figure 2 DIDS (a RAD51 inhibitor Figure 2 A- Figure 2 D), B02( Figure 2 E-F) Graphs representing the results of inhibition of PRV replication in A549 cells.

[0019] Figure 3 This is a characterization of the results of the RAD51 inhibitor B02 inhibiting the replication of pseudorabies virus in PK-15 cells. DETAILED DESCRIPTION

[0020] The present invention will be described in further detail below with reference to the accompanying drawings and examples. It is particularly noted that the following examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. Similarly, the following examples are only some embodiments of the present invention and are not intended to be exhaustive. All other embodiments obtained by those of ordinary skill in the art without creative effort are intended to fall within the scope of protection of the present invention.

[0021] It should also be noted here that in order to avoid obscuring the technical solution of the patent of the present invention due to unnecessary details, only the technical solutions and / or processing steps closely related to the solution of the patent of the present invention are shown in the embodiments, and other details that are not very relevant are omitted.

[0022] The basic information of the RAD51 inhibitor used in the following example scheme is as follows:

[0023] Inhibitor DIDS: MCE, product number HY-D0086;

[0024] Inhibitor B02: MCE, product number HY-101462.

[0025] Example 1

[0026] Cytotoxicity of RAD51 inhibitors DIDS and B02 in A549 cells

[0027] 1. Experimental methods

[0028] The small molecule RAD51 inhibitors DIDS and B02 were dissolved in dimethyl sulfoxide (DMSO) solvent, stored in a -20°C refrigerator after aliquoting, and diluted to the working concentration with the corresponding cell maintenance medium before use.

[0029] A549 cells were cultured at 1×10 4 Cells were seeded at 100 μg / well in a 96-well plate. When the cell density reached 70%, the culture medium was discarded and the inhibitors DIDS and B02 were diluted to six concentrations (from low to high): 1 μM, 2.5 μM, 5 μM, 10 μM, 20 μM, and 40 μM) in high-glucose DMEM (containing 2% FBS, penicillin 100 U / mL, streptomycin 50 μg / mL). Six replicate wells were prepared for each concentration.

[0030] After incubating A549 cells with inhibitors DIDS and B02 in a 37°C, 5% CO2 incubator for 12 hours, 10% CCK8 reagent (10 μL / well) was added to each well in the dark. After further incubation for 1 hour in the dark, the cells were shaken at low speed for 1 minute. The absorbance of each well was read at a reference wavelength of 450 nm using a microplate reader. The cell viability was calculated using the formula using the detected OD (450 nm) value, and then analyzed using GraphPad Prism software.

[0031] The cell survival rate was calculated as follows: (OD value of the experimental group - OD value of the blank group) / (OD value of the control group - OD value of the blank group) × 100%.

[0032] 2. Experimental results

[0033] The results are as follows Figure 1 As shown in A, the RAD51 inhibitor DIDS showed no significant cytotoxicity in A549 cells when treated with it in the concentration range of 1-40 μM; Figure 1 As shown in B, the RAD51 inhibitor B02 had no significant effect on cell viability at concentrations of 1 μM, 2.5 μM, 5 μM, 10 μM, and 20 μM, but showed certain cytotoxicity at 40 μM (*P<0.05).

[0034] Example 2

[0035] RAD51 inhibitors DIDS and B02 inhibit pseudorabies virus replication in A549 cells

[0036] 1. Experimental methods

[0037] A549 cells were cultured at 1×10 6Cells were seeded at 400 μg / well in a 6-well plate. When the cell density reached 80%, the culture medium in the plate was discarded and replaced with DMEM medium containing 2% FBS containing 5 μM and 10 μM DIDS and B02. After pre-incubation for 1 hour in a 37°C, 5% CO2 incubator, the cells were infected with PRV at an MOI of 0.1. After 1 hour of infection, the virus solution was discarded, the cells were washed three times with PBS, and the cells were replaced with DMEM medium containing 2% FBS containing the above-mentioned corresponding concentrations of DIDS and B02 and incubated for another 12 hours. RNA extraction, reverse transcription, RT-PCR and RT-qPCR steps were then performed in sequence. RT-PCR and RT-qPCR were used to detect the effects of small molecule compounds DIDS and B02 on PRV gE and gD gene transcription.

[0038] 2. Experimental results

[0039] The changes in the expression levels of viral genes gE and gD were detected by RT-PCR and RT-qPCR. The results showed that compared with the control group, 5μM DIDS treatment of A549 cells significantly reduced the transcription levels of PRV gE and gD mRNA ( Figure 2 A, Figure 2 B). In addition, treatment of A549 cells with 10 μM DIDS also significantly inhibited the replication of PRV ( Figure 2 C, Figure 2 D). RT-PCR detection of gE and gD expression after PRV infection showed that the expression levels of gE and gD mRNA in the B02 treatment group (5μM and 10μM) were significantly lower than those in the control group ( Figure 2 E, Figure 2 F) These results indicate that the RDA51 inhibitors DIDS and B02 inhibit the transcription of PRV gE and gD genes in A549 cells. This suggests that both DIDS and B02 can inhibit PRV replication.

[0040] Example 3

[0041] RAD51 inhibitor B02 inhibits pseudorabies virus replication in PK-15 cells

[0042] 1. Experimental methods

[0043] PK-15 cells were cultured at a rate of 1×10 6Cells were seeded at 100 μg / well in a 6-well plate. When the cell density reached 80%, the culture medium in the plate was discarded and replaced with 2% FBSDMEM medium containing 5 μM and 10 μM B02. After pre-incubation for 1 hour in a 37°C, 5% CO2 incubator, the cells were infected with PRV at an MOI of 0.1. After 1 hour of infection, the virus solution was discarded, the cells were washed three times with PBS, and the culture medium was replaced with 2% FBS DMEM medium containing the above-mentioned corresponding concentration of B02 and incubated for another 12 hours. RNA extraction, reverse transcription, and RT-PCR were then performed in sequence. RT-PCR was used to detect the effect of B02 on the transcription of PRV gE and gD genes.

[0044] 2. Experimental results

[0045] RT-PCR assays were performed to detect the expression of gE and gD genes after PRV infection. Treatment with 5 μM and 10 μM B02 significantly inhibited PRV replication in PK-15 cells. These results indicate that the RDA51 inhibitor B02 inhibits the transcription of PRV gE and gD genes in PK-15 cells.

[0046] Figure 3 This is a characterization of the results of the RAD51 inhibitor B02 inhibiting the replication of pseudorabies virus in PK-15 cells.

[0047] Based on the results of the above three examples, it can be demonstrated that the RAD51 inhibitors DIDS and B02 have the effect of inhibiting pseudorabies virus replication in A549 cells, and the RAD51 inhibitor B02 also has the effect of inhibiting pseudorabies virus replication in PK-15 cells. Therefore, the RAD51 inhibitors DIDS and B02 can be used as small molecule compounds to prepare anti-pseudorabies virus infection drugs.

[0048] The above descriptions are only some embodiments of the present invention and do not limit the scope of protection of the present invention. Any equivalent device or equivalent process transformation made by using the contents of the description and drawings of the present invention, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. Application of RAD51 inhibitors in the preparation of anti-pseudorabies virus drugs.

2. The use according to claim 1, characterized in that: The RAD51 inhibitor is the RAD51 inhibitor DIDS and / or the RAD51 inhibitor B02.

3. Application of RAD51 inhibitors in the preparation of drugs for inhibiting the replication of pseudorabies virus in cells.

4. The use according to claim 3, characterized in that: The RAD51 inhibitor is the RAD51 inhibitor DIDS and / or the RAD51 inhibitor B02.

5. The use according to claim 4 or 5, characterized in that: The cells are A549 cells and / or PK-15 cells.

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