Application of CDK4 / 6 inhibitor in preparation of drug for resisting DNA virus

Abstract

The invention discloses application of a CDK4 / 6 inhibitor in preparation of drugs for resisting DNA viruses, and relates to the field of biological medicines. Through small molecule inhibitor screening work of a DNA virus HSV-1 cell infection model, it is found that various targeted CDK4 / 6 inhibitors can effectively inhibit HSV-1 (GFP) cell infection. By constructing a CDK4 knockout cell line, it is definitely limited that CDK4 can enhance natural immune signals, and therefore the DNA virus resistance is enhanced. According to the invention, an HSV-1 mouse eye infection model is utilized, and the application advantages of the whole animal level CDK4 / 6 inhibitor in the preparation of antiviral drugs are explored.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to the use of a CDK4 / 6 inhibitor in the preparation of a drug for resisting DNA viruses. Background Art

[0002] Cyclin-dependent kinase 4 / 6 (CDK4 / 6) is a core molecule for cell cycle regulation. It drives the G1 / S phase transition of the cell cycle by phosphorylating retinoblastoma protein (Rb) and is a cell cycle targeted drug for treating tumors ([1] Goel S, Bergholz JS, Zhao JJ. Targeting CDK4 and CDK6 in cancer. Nat Rev Cancer. 2022; 22(6): 356-72. [2] Fass1 A, Geng Y, Sicinski P. CDK4 and CDK6 kinases: From basic science to cancer therapy. Science (New York, NY). 2022; 375(6577): eabc1495.). The CDK4 / 6 inhibitors that have been marketed, such as palbociclib and abemaciclib, have shown significant clinical advantages in the treatment of metastatic breast cancer by specifically blocking the CDK4 / 6-Rb-E2F signaling pathway. It can not only selectively inhibit the proliferation of tumor cells, but also because of its good pharmacokinetic properties and controllable safety characteristics (the main adverse reaction is reversible neutropenia), it is used in combination drug treatment for cancer ([1] Goodwin CM, Waters AM, Klomp JE, et al. Combination Therapies with CDK4 / 6 Inhibitors to Treat KRAS-Mutant Pancreatic Cancer. Cancer Res. 2023; 83(1): 141-57. [2] O'Leary B, Finn RS, Turner NC. Treating cancer with selective CDK4 / 6 inhibitors. Nature reviews Clinicaloncology. 2016; 13(7): 417-30.).

[0003] Recent studies have found that CDK4 / 6 inhibitors have shown great potential in the field of immune regulation. They can reshape the tumor immune microenvironment by inhibiting the proliferation of regulatory T cells and enhancing antigen presentation, thereby promoting the recognition and clearance of tumor cells by immune cells and significantly enhancing the body's immune surveillance function ([1] Goel S, DeCristo MJ, Watt AC, et al. CDK4 / 6 inhibition triggers anti-tumour immunity. Nature. 2017; 548(7668): 471-5. [2] Deng J, Wang ES, Jenkins RW, et al. CDK4 / 6 Inhibition Augments Antitumor Immunity by Enhancing T-cell Activation. Cancer discovery. 2018; 8(2): 216-33.). However, in terms of antiviral host defense, the function and mechanism of action of CDK4 / 6 inhibitors have not been fully studied.

[0004] DNA viruses are a class of pathogens with important clinical significance, mainly including herpesviridae (such as herpes simplex virus HSV-1 / 2, varicella-zoster virus VZV), poxviridae (such as vaccinia virus VacV), papillomaviridae (such as human papillomavirus HPV) and hepadnaviridae (such as hepatitis B virus HBV). These viruses can cause a wide spectrum of diseases, from acute infectious diseases (such as chickenpox and herpes zoster) to chronic persistent infections (such as HBV chronic hepatitis), and are even closely related to the occurrence of various malignant tumors (such as cervical cancer caused by high-risk HPV infection and hepatocellular carcinoma related to HBV infection).

[0005] Currently, commonly used antiviral drugs (such as nucleoside / nucleotide analogs) in clinical practice mainly limit viral genome replication by inhibiting viral DNA polymerase activity. However, this type of drug has limitations: on the one hand, the virus can develop drug resistance through gene mutations, such as lamivudine resistance caused by HBV polymerase rtM204V / I mutations; on the other hand, long-term use of drugs may have drug side effects (such as high concentrations of tenofovir accumulated in renal tubular epithelial cells increase the risk of renal tubular damage).

[0006] Host-oriented antiviral strategies not only have broad-spectrum antiviral potential by regulating the host's inherent immune response (such as activating the DNA recognition signaling pathway cGAS-STING) or targeting host factors that viral replication depends on, but also can effectively reduce the risk of viral resistance, providing a new direction for antiviral treatment. In the prior art, for example, the patent application with publication number CN115920006A discloses the use of ABCF1 or its agonists in the preparation of anti-DNA virus preparations. The invention study verifies that the new cccDNA interaction protein ABCF1 (ATP-binding cassette sub-family Fmember 1) can bind to cccDNA and inhibit cccDNA transcription by phase separation, thereby inhibiting the application of HBV replication, and further clarifies that the N-terminal domain of ABCF1 plays a key role in binding to cccDNA, and provides the application of ABCF1 or its agonists in the development of anti-HBV infection related drugs. And the patent application with publication number CN118178652A discloses a method for treating hepatitis B virus infection by administering NS5A inhibitors, NS5B inhibitors, NS3 inhibitors or a combination thereof.

[0007] Therefore, exploring new antiviral strategies and drugs, especially those compounds that can enhance the host's own antiviral capabilities, is of great significance for improving treatment outcomes and preventing viral diseases. Summary of the invention

[0008] Based on the deficiencies of anti-DNA virus therapeutic drugs in the prior art, the present invention provides a new drug application strategy: CDK4 / 6 inhibitors (such as palbociclib, abemaciclib, etc.) approved by the FDA for the treatment of breast cancer are applied to the preparation of anti-DNA virus drugs. Based on the cell model and mouse model infected with DNA viruses, the present invention develops and optimizes the application scheme of CDK4 / 6 inhibitors in the preparation of antiviral drugs, aiming to provide a new broad-spectrum and highly effective antiviral treatment method by enhancing the host's inherent immune defense capabilities.

[0009] Based on the previous screening of small molecule inhibitors for herpes simplex virus type 1 (HSV-1) cell infection models, the applicant found that a variety of small molecule inhibitors targeting CDK4 can effectively inhibit HSV-1 viral infection. Taking into account the target specificity, clinical safety and pharmacokinetic properties of the drug, the present invention prefers CDK4 / 6 inhibitors palbociclib and abemaciclib for further study. By optimizing the dosing concentration and time window, combined with HSV-1GFP infected cell experiments and mouse eye infection models, it was confirmed that palbociclib and abemaciclib have significant anti-HSV-1 activity both in vitro and in vivo.

[0010] Further mechanism studies have shown that CDK4 / 6 inhibitors significantly enhance the intensity of host cell's antiviral innate immune response by enhancing the levels of downstream interferon-related genes in the innate immune cGAS-STING signaling pathway. Specifically, in the CDK4 knockout cell line, the mRNA levels of IFIT1 and ISG15, the downstream genes of the cGAS-STING pathway, were significantly enhanced, while the viral load of DNA viruses HSV-1 and VacV was significantly reduced. Since the cGAS-STING pathway can recognize abnormal DNA in the cytoplasm (such as the viral genome released into the cytoplasm during DNA virus infection), the present invention speculates that the antiviral effect of CDK4 / 6 inhibitors has a broad spectrum and can be applied to the treatment of a variety of DNA virus infections, including but not limited to herpes simplex virus (HSV), vaccinia virus (VacV), human papillomavirus (HPV) and hepatitis B virus (HBV).

[0011] The specific technical solutions of the present invention are as follows:

[0012] The present invention provides the use of a CDK4 / 6 inhibitor in the preparation of a drug for resisting DNA viruses. In combination with a CDK4 knockout cell line, it is clear that inhibiting CDK4 / 6 can enhance antiviral host defense, thereby inhibiting DNA virus infection.

[0013] Preferably, the CDK4 / 6 inhibitor is at least one of palbociclib and abemaciclib.

[0014] Preferably, when used, the dosage of palbociclib in the drug is 1-10 micromoles; or the dosage of abemaciclib in the drug is 0.1-1 micromoles.

[0015] Preferably, the DNA virus is at least one of herpes simplex virus, vaccinia virus, human papillomavirus, and hepatitis B virus.

[0016] More preferably, the herpes simplex virus is herpes simplex virus type 1.

[0017] Preferably, the drug is in the form of tablets, capsules, granules, oral liquid, pills, aerosol or injection.

[0018] The present invention also provides a drug for resisting DNA viruses, wherein the active ingredient of the drug comprises a CDK4 / 6 inhibitor, and the CDK4 / 6 inhibitor is at least one of palbociclib and abemaciclib.

[0019] Preferably, the DNA virus is at least one of herpes simplex virus, vaccinia virus, human papillomavirus and hepatitis B virus.

[0020] More preferably, the herpes simplex virus is herpes simplex virus type 1.

[0021] Beneficial effects of the present invention:

[0022] The present invention screened small molecule inhibitors in a cell infection model of the DNA virus HSV-1 (herpes simplex virus type 1) and found that a variety of inhibitors targeting CDK4 can effectively inhibit HSV-1 cell infection, including CDK4 / 6 inhibitors palbociclib and abemaciclib.

[0023] The present invention constructs a CDK4 knockout cell line, and clearly shows that inhibiting CDK4 can enhance host defense against DNA viruses.

[0024] The present invention utilizes the HSV-1 mouse eye infection model to explore the application advantages of CDK4 / 6 inhibitors in the preparation of antiviral drugs at the whole animal level. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Results of screening small molecule inhibitors of cell cycle kinases related to HSV-1 virus.

[0026] Figure 2 This is a diagram of the results of a DNA virus (HSV-1GFP) cell infection experiment. Green fluorescence indicates virus infection, and the scale bar is 100 microns.

[0027] Figure 3 The infection levels of DNA viruses HSV-1 and VacV in wild-type and CDK4 knockout DLD1 cells were detected by viral mRNA levels; *** indicates p < 0.001.

[0028] Figure 4 The results of mRNA levels of innate immune response-related proteins IFIT1 and ISG15 in wild-type and CDK4-knockout DLD1 cells; *** indicates p < 0.001.

[0029] Figure 5 This is a diagram of the mechanism by which the innate immune cGAS-STING pathway of the present invention recognizes abnormal dsDNA in the cell cytoplasm.

[0030] Figure 6 The disease score results of corneal infection in mice and the severity of eye infection after CDK4 / 6 inhibitor intervention are shown in the figure; ** indicates p<0.01, *** indicates p<0.001.

[0031] Figure 7The HSV-1 viral load in the eyeballs and eyelids of HSV-1 corneal-infected mice on the 6th day after intervention with the CDK4 / 6 inhibitors palbociclib and abemaciclib (A) and the mRNA expression of innate immune response-related proteins ISG15 and CXCL10 on the 3rd day (B); * indicates p<0.05, ** indicates p<0.01, and *** indicates p<0.001. DETAILED DESCRIPTION

[0032] Example 1

[0033] DLD1 cells were pretreated with a variety of cell cycle kinase-related small molecule inhibitors (all at 1 micromolar working concentration) for 16 hours, and then HSV-1 (luciferase) was used for DNA virus cell infection experiments. Cells were collected and lysed 8 hours after infection, and the relative viral load was obtained through luciferase substrate reaction. Figure 1 As shown, the results showed that 8 of the 22 cell cycle kinase-related small molecule drugs had the effect of effectively reducing the amount of HSV-1 (luciferase) virus (reduced by more than 50%). Analysis of these 8 small molecules found that the targets of 6 inhibitors included CDK4 and the substrates of 5 included CDK6. Since there are no single small molecule inhibitors of CDK4 or CDK6, we selected CDK4 / 6 specific drugs with better specificity and reported lower side effects for subsequent experiments.

[0034] Example 2

[0035] In the DNA virus cell infection experiment using HSV-1 (GFP) in DLD1 cells, green fluorescence (GFP) can be observed to visually indicate the level of virus infection.

[0036] In the control group using DMSO as solvent, 1 micromolar Palbociclib was treated for 12 hours, or 100 nanomolar Abemaciclib was treated for 12 hours, and glHSV-1GFP virus was added to infect the cells, and the cells were photographed 12 hours after infection. Figure 2 As shown, CDK4 / 6 inhibitors palbociclib and abemaciclib can effectively inhibit cell infection of glHSV-1GFP virus.

[0037] Example 3

[0038] CRISPR / Cas9 technology was used to knock out CDK4, one of the direct acting proteins of CDK4 / 6 inhibitors, in DLD1 cells. The sgRNA primers for human CDK4 were designed and synthesized, and the nucleotide sequence is as follows:

[0039] Forward primer: 5-'CACCGgagaacattctggtgacaag-3';

[0040] Reverse primer: 5-'AAACcttgtcaccagaatgttctcC-3'.

[0041] The primers were cloned into the expression vector pX330, and the recombinant plasmid was obtained by bacterial transformation and plasmid extraction, and then sequenced and verified. DLD1 cells were cultured to ensure that the cells were in good growth state, and the recombinant plasmid and the plasmid expressing Cas9-GFP were transfected into DLD1 cells. After 36 hours, GFP-positive flow cytometry screening was performed to obtain monoclonal cells. Total protein was extracted from the screened cells, and the CDK4-knockout DLD1 cell line was obtained by using CDK4-specific antibodies and immunoprecipitation experiments.

[0042] In DLD1 wild-type and CDK4 knockout cell lines, cells were infected with two DNA viruses, HSV-1 and VacV, respectively. Cells were collected 8 hours after infection, and the ratio of viral mRNA to the mRNA of the cellular reference gene was used to analyze the virus infection level. Figure 3 As shown, the results showed that in cells with CDK4 knockout, the viral load was significantly reduced. This shows that knocking out CDK4 can effectively reduce the cell infection of the two DNA viruses HSV-1 and VacV.

[0043] The mRNA levels of interferon-related proteins IFIT1 and ISG15 in cells were also detected. Figure 4 As shown in the results, the mRNA levels of IFIT1 and ISG15, which are downstream of the innate immune cGAS-STING pathway in cells, were significantly increased in CDK4 knockout cells. This shows that limiting CDK4 can effectively enhance the host's innate immune response. According to this part of the mechanism, Figure 5 As shown, the innate immune cGAS-STING pathway recognizes abnormal dsDNA in the cell cytoplasm (including dsDNA of viral origin), so the antiviral function targeting CDK4 can be applied to the preparation of antiviral drugs for a variety of DNA viruses.

[0044] Example 4

[0045] At the whole animal level, HSV-1 virus was dripped into the eyes of BalB / C mice to simulate corneal infection. The virus titer was 7.54×10 6pfu, on Day 0, take 1 microliter of the virus stock solution and dilute it to 5 microliters with cell culture medium. After anesthetizing the mouse, drip the virus to infect the left eye of the mouse. The dilution is performed to ensure the infection efficiency of the virus and prevent the virus from being too concentrated in a certain location. Serum-free cell culture medium DMEM is dripped into the right eye as a blank experimental control. In the next 4 days, 1 microliter of Palbocilib 10 micromoles and Abemaciclib 1 micromoles are used every day, diluted to 10 microliters with cell culture medium, and administered after anesthetizing the mouse. On the 5th day, only observation is done without any treatment. Because the mouse eye socket is small, about 5 microliters are dripped each time, and the remaining 5 microliters are added after waiting for 10-15 minutes. The purpose of dilution is to avoid uneven contact between the drug and the mouse eye, and to ensure that a certain amount of drug stays in the mouse eye. On the 3rd day, disease scoring of all mouse eye infections was performed ( Figure 6 ), and the eyelid and eyeball samples of 4 mice in each group were taken to measure the mRNA levels of interferon-related genes ISG15 and CXCL10 in natural immune signals for analysis and comparison ( Figure 7 B) On the 6th day, the eyes of the mice were photographed and the disease scores were calculated according to the severity of the eye infection. Figure 6 As shown, the results showed that mice treated with CDK4 / 6 inhibition had a significant improvement in ocular infection.

[0046] The eyeballs and eyelids of mice were taken on the 6th day to detect the viral load of HSV-1. Figure 7 As shown in A, the results showed that after administration of CDK4 / 6 inhibitors Palbociclib or Abemaciclib, the amount of HSV-1 virus in mouse tissue samples was significantly reduced, and the genes ISG15 and CXCL10 related to the innate immune response were significantly upregulated on the third day of infection administration ( Figure 7 B). This indicates that CDK4 / 6 inhibitors have the potential to be used in the preparation of drugs against DNA viruses.

Claims

1. Application of CDK4 / 6 inhibitors in the preparation of drugs for anti-DNA viruses.

2. The use according to claim 1, characterized in that: The CDK4 / 6 inhibitor is at least one of palbociclib and abemaciclib.

3. The use according to claim 2, characterized in that: When used, the dosage of palbociclib in the drug is 1-10 micromoles; or the dosage of abemaciclib in the drug is 0.1-1 micromoles.

4. The use according to claim 1, characterized in that: The DNA virus is at least one of herpes simplex virus, vaccinia virus, human papillomavirus and hepatitis B virus.

5. The use according to claim 4, characterized in that: The herpes simplex virus is herpes simplex virus type 1.

6. The use according to claim 1, characterized in that: The dosage form of the medicine is tablet, capsule, granule, oral liquid, dripping pill, aerosol or injection.

7. A drug for resisting DNA virus, characterized in that: The active ingredient of the drug contains a CDK4 / 6 inhibitor, and the CDK4 / 6 inhibitor is at least one of palbociclib and abemaciclib.

8. The drug for resisting DNA virus according to claim 7, characterized in that: The DNA virus is at least one of herpes simplex virus, vaccinia virus, human papillomavirus, hepatitis B virus and hepatitis E virus.

9. The drug for resisting DNA virus according to claim 8, characterized in that: The herpes simplex virus is herpes simplex virus type 1.

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