Method for rapidly screening kinase inhibitor resistant to Zika virus infection and application

Abstract

The invention provides a method for rapidly screening a Zika virus infection resistant kinase inhibitor and application. The method is used for identifying a host kinase target spot for regulating and controlling Zika virus (ZIKV) infection. Based on the obtained kinase target, a rapid verification and kinase inhibitor development platform is further established, and a new strategy is provided for research and development of anti-ZIKV drugs of targeted host factors. According to the kinase target, the key dependent host kinase replicated by ZIKV is systematically identified by constructing kinase specific sgRNA library cells A549 and combining high-throughput function screening. The method overcomes the limitation that a traditional virus targeted drug easily generates drug resistance, realizes efficient conversion from host target discovery to potential inhibitor development, and solves the key problems of low flux, long period and high false positive rate in the prior art.

Description

Technical Field

[0001] This invention belongs to the field of antiviral drug screening technology, specifically relating to a method for rapidly screening kinase inhibitors against Zika virus infection. Background Technology

[0002] Zika virus (ZIKV) is a mosquito-borne member of the Flavivirus genus. Listed as a high-risk pathogen by the World Health Organization (WHO), it has been shown to cause severe neurodevelopmental disorders since its large-scale outbreak in Brazil in 2015, including microcephaly and Guillain-Barré syndrome (GBS) (PMID: 27028561; PMID: 26948433). Furthermore, ZIKV infection is also associated with neurological damage and male reproductive system impairment in adults (PMID: 27798603). Although ZIKV, along with Ebola and Nipah viruses, is a pandemic threat, there are currently no approved vaccines or specific antiviral drugs globally (PMID: 30158602), and clinical treatment remains primarily symptomatic.

[0003] Current antiviral research on ZIKV mainly focuses on two strategies: (1) direct targeting of viral proteins. However, the high mutation rate of viral RNA polymerase easily leads to drug resistance, and some inhibitors have limited inhibitory effects on ZIKV; (2) host-targeted therapy: disrupting the cellular environment dependent on viral replication by regulating host factors (such as kinases and ubiquitin-proteasome system) (PMID: 30149598). Such strategies can reduce the risk of drug resistance, but the selectivity and safety of key host targets remain challenges. Existing methods (such as RNA interference) have significant off-target effects, resulting in a high false positive rate. Emerging technologies such as CRISPR-Cas9 have not been widely applied to anti-ZIKV research, resulting in a lag in the systematic screening of host kinase targets; in addition, the development cycle of new compounds is long, making it difficult to cope with sudden outbreaks; target discovery and inhibitor development are disconnected, lacking a rapid matching translation platform; the antiviral potential of approved kinase inhibitors has not been systematically evaluated.

[0004] Therefore, there is an urgent need for a method to quickly screen kinase inhibitors that resist Zika virus infection, providing new ideas for target selection, prioritizing kinase inhibitors that have passed safety tests (such as SNS-032), and shortening the research and development cycle. Summary of the Invention

[0005] To address or partially address the problems existing in related technologies, this application provides a method for rapidly screening kinase inhibitors against Zika virus (ZIKV) infection. The method identifies host kinase targets against ZIKV infection using a CRISPR-Cas9 kinase library and verifies the antiviral activity of its inhibitors. This method is used to rapidly identify host kinase targets against ZIKV infection and verify the antiviral activity of its inhibitors.

[0006] This application provides a method for rapidly screening kinase inhibitors against Zika virus infection, comprising the following steps: (1) Construct a stable cell bank for transfection; (2) After the stable transfection cell bank in (1) is constructed, the cells are inoculated with ZIKV SZ-WIV01 strain. After the cytopathic effect appears, the sgRNA region is amplified by PCR and high-throughput sequencing analysis is performed. (3) Compare the differences in sgRNA abundance between the infected group and the control group, screen for sgRNAs that are significantly missing in the infected cells, and sort the candidate genes to obtain the kinase target against Zika virus infection; the kinase target is CDK2.

[0007] Furthermore, the MOI of the ZIKV SZ-WIV01 strain is 1.

[0008] Furthermore, the cell library is a CRISPR-Cas9 kinase knockout library of cells, A549.

[0009] Furthermore, in step (3), the MAGeCK algorithm is used to compare the difference in sgRNA abundance between the infected group and the control group.

[0010] Furthermore, in (3), candidate genes are ranked based on statistical significance.

[0011] This application also provides the use of a screened CDK2 inhibitor in the preparation of an anti-ZIKV drug, wherein the use does not involve the process of preventing or treating the disease.

[0012] Beneficial effects

[0013] (1) This application focuses on kinase targets and is specifically designed for the human kinase genome, which significantly improves the screening accuracy and greatly reduces interference from unnecessary genes compared to whole-genome libraries. Multiple host kinases regulate viral protein function through phosphorylation modification, providing a theoretical basis for intervention strategies targeting host kinases. Kinase families have unique advantages in drug development; their conserved ATP-binding pockets facilitate drug design, their activity can be directly regulated by small molecule compounds, and their clinical translation potential has been fully validated. This method, by focusing on kinase genome screening, effectively eliminates interference from genes unrelated to the viral life cycle in terms of specificity, making sequencing data more reliable; in terms of sensitivity, it significantly improves the coverage depth of target kinase genes at the same sequencing throughput; and in terms of practicality, the screening results directly point to kinase targets with clear drug development potential, laying a solid foundation for subsequent inhibitor development.

[0014] (2) An integrated "infection-screening-validation" process was established, effectively solving the following technical problems: traditional host factor screening has low throughput and long cycle; a single experimental system is difficult to take into account both target discovery and functional validation; and the technical defects of insufficient validation of the correlation between host targets and antiviral activity. Through the construction of kinase-specific sgRNA libraries and high-throughput infection models, the regulatory role of kinase gene knockout on viral replication was systematically simulated; based on the molecular correlation between kinase activity and key steps in the viral life cycle (such as endocytosis, replication, assembly, etc.), a multi-dimensional validation system was established; and the kinase-inhibitor interaction network theory was used to achieve rapid transformation from target discovery to potential inhibitor development. This technology platform provides a new strategy for developing anti-ZIKV drugs that target host factors, overcoming the limitation of traditional viral targeted drugs being prone to drug resistance.

[0015] (3) In vitro, viral RNA (qPCR), protein (WB), and viral titer levels in the supernatant were simultaneously detected in cells. In vivo, the viral infection status and inflammation level were monitored using an immunodeficient mouse model with type I interferon receptor knockout. Traditional immunocompetent animal models often mask the true pathogenicity of the virus and the effect of drug intervention due to the rapid antiviral response of the host's type I interferon system, leading to biased drug evaluation results. The establishment of this model is based on the following theoretical basis: the type I interferon (IFN-α / β) signaling pathway is the core mechanism of host antiviral immunity, and its receptor deletion can make mice susceptible to Zika virus; at the same time, this model retains the complete inflammatory response pathway, which can truly reflect the cytokine storm (such as increased levels of IL-6 and TNF-α) and histopathological damage caused by viral infection. By systematically monitoring the dynamic changes of viral replication (qPCR detection) and the levels of inflammatory factors in this model, the dual effects of candidate drugs in inhibiting viral replication and alleviating inflammatory damage can be evaluated simultaneously.

[0016] (4) Solve the problem of long conversion cycle: compounds in the clinical development stage (such as SNS-032).

[0017] (5) The solution provided in this application achieves efficient screening and rapid verification of anti-ZIKV host targets through the above-mentioned innovative design, and solves the key problems of low throughput, long cycle and high false positive rate in the prior art.

[0018] Highly efficient screening: The CRISPR-Cas9 kinase library screening technology used in this invention has significant high-throughput advantages, enabling rapid and systematic identification of host kinase targets. Compared to traditional RNA interference technology (which typically requires 4-6 weeks to complete whole-genome screening) and small molecule library screening (requiring multiple rounds of validation and taking 2-3 months), this method significantly improves screening efficiency and accuracy through optimized sgRNA design and a dual-parameter evaluation system (viral inhibition rate + cell viability). Screening results can be directly integrated into clinical-stage kinase inhibitor databases, enabling rapid translation from target discovery to therapeutic strategies. Target inhibitor selection: SNS-032 and Fadraciclib have passed phase I / II clinical trials (cancer indication). This invention innovatively discovers their anti-ZIKV use, and the effective dose (1-5 μM) is far lower than the maximum tolerated clinical dose (40 mg / m²). 2 ).

[0019] It should be understood that the above general description and the following detailed description are exemplary and explanatory only, and do not limit this application. Attached Figure Description

[0020] The above and other objects, features and advantages of this application will become more apparent from the more detailed description of exemplary embodiments of this application taken in conjunction with the accompanying drawings, wherein the same reference numerals generally represent the same components in the exemplary embodiments of this application.

[0021] Figure 1Figure A shows the screening of CRISPR / Cas9 kinase libraries to identify CDK2 as a ZIKV infection-dependent factor. Figure B is a schematic diagram of CRISPR / Cas9 kinase library screening used to identify host factors required for Zika virus (ZIKV) replication. A549 cells were transduced with a lentiviral mCherry-sgRNA library targeting 1,098 human kinase genes. After Zika virus infection (MOI = 1), DNA was extracted and deep sequenced from surviving cells to identify enriched gene targets. Figure B shows the targeted overexpression (OE) of CDK2 in A549 cells via lentiviral transduction, followed by ZIKV infection (MOI = 0.5), and analysis was performed by qPCR. The figure shows the levels of the overexpressed gene transcript CDK2 and ZIKV NS5 RNA. Figure C shows the Western blot (WB) analysis of relative protein expression in CDK2-overexpressing A549 cells, and the protein levels (CDK2 / ZIKV) were quantified using ImageJ software. Figure D shows the qPCR analysis of the overexpressed gene transcripts (CDK2 mRNA) and ZIKV RNA levels in CaCo2 cells overexpressing CDK2. Figure E shows the Western blotting analysis of CDK2 and ZIKV NS3 / NS5 protein levels in CaCo2 cells overexpressing CDK2. Protein levels were quantified using ImageJ software.

[0022] Figure 2 Figure A shows the in vitro antiviral activity (CDK2 inhibitors exhibited significant anti-Zika virus activity in vitro); Figure A shows the antiviral activity and cytotoxicity of SNS-032. ZIKV particle levels in the supernatant after treatment with different concentration gradients of SNS-032 were quantitatively detected by qPCR, and parallel cytotoxicity in uninfected cells was assessed using the CCK-8 assay. Key parameters included: EC50. 50 (Hardest effective concentration for inhibiting ZIKV), CC 50 (Half-maximal cytotoxicity concentration) and SI (selectivity index, i.e., CC) 50 / EC 50Figure B shows the antiviral activity and cytotoxicity of Fadraciclib. As shown in section (A), Zika virus (ZIKV) particle production and cell viability were evaluated at different concentrations of Fadraciclib gradients. Figure C shows the inhibitory effect of SNS-032 on Zika virus (ZIKV) protein expression. A549 and CaCo2 cells were pretreated with DMSO (control) or 2 µM SNS-032, followed by Zika virus infection or simulated infection (PBS). CDK2 and ZIKV protein levels were analyzed by Western blot (WB), and band intensity was quantified using ImageJ software. Figure D shows the dose-dependent inhibition of ZIKV protein expression. A549 cells were infected with ZIKV after treatment with SNS-032 or Fadraciclib (0–3 µM). The expression of viral capsid protein (C) and NS5 protein was detected by Western blot (WB), and quantitative analysis was performed using ImageJ. Figure E is a schematic diagram of the CDK2 inhibitor treatment regimen, showing the time axis of inhibitor administration relative to Zika virus (ZIKV) infection: pretreatment (−2 h), cotreatment (0 h), or posttreatment (+2 h). Figure F shows the time-dependent antiviral effect of SNS-032. A549 cells were treated with 2 µM SNS-032 before, during, or after infection. The levels of ZIKV NS5 protein (Western blotting, left) and mRNA (real-time quantitative PCR, right) were analyzed. Figure G shows the time-dependent antiviral effect of Fadraciclib. Similar to group (F), 2 µM Fadraciclib was used, and the levels of Zika virus NS5 protein and mRNA were detected by Western blotting (WB) and real-time quantitative PCR (qPCR), respectively. In the figures, CYC065 is an abbreviation for Fadraciclib.

[0023] Figure 3 This is an in vivo evaluation of the anti-Zika virus infection effect of CDK2 inhibitors (reduction of viral RNA levels and improvement of inflammation); Figure A is a schematic diagram of the in vivo anti-Zika virus (ZIKV) assessment experimental design. Five-week-old A129 mice (n=4-5 per group) were infected with the virus via intraperitoneal injection (ip) on day 0. 5Zika virus was injected with PFU daily, followed by intraperitoneal injections of SNS-032 (10 mg / kg), Fadraciclib (15 mg / kg), temopofen (0.5 mg / kg; positive control), or a solvent (DMSO; negative control) for five consecutive days. Body weight was monitored daily, and blood was collected every two days via tail vein until day 9, at which point mice were sacrificed for analysis of blood, liver, and brain tissue. Figure B shows the percentage change in body weight relative to initial body weight, illustrating the treatment effect over time. Figure C shows the viral RNA level in whole blood determined by qPCR, and Figures D and E show the relative ZIKV mRNA levels in brain and liver tissue. Figure F shows the expression of ZIKV NS5 protein in brain and liver tissue analyzed by Western blot, with quantification of band intensity using ImageJ software.

[0024] Figure 4 The CDK2 inhibitor exhibits broad-spectrum anti-DENV activity (WB data from four serotypes indicate that the CDK2 inhibitor demonstrates broad-spectrum anti-dengue virus activity in vitro). A549 cells were pretreated with SNS-032 or Fadraciclib (0-3 μM concentration gradient) for 4 hours one hour before infection with DENV serotypes 1-4. After virus adsorption, cells were maintained in inhibitor-containing medium for 48 hpi. Western blot analysis showed dose-dependent inhibition of DENV protein expression in all four serotypes: AB plots represent DENV-1, CD plots represent DENV-2, EF plots represent DENV-3, and GH plots represent DENV-4 (WB results). Detailed Implementation

[0025] Embodiments of this application will now be described in more detail with reference to the accompanying drawings. While embodiments of this application are shown in the drawings, it should be understood that this application may be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided to make this application more thorough and complete, and to fully convey the scope of this application to those skilled in the art.

[0026] All experimental materials used below were purchased from the market.

[0027] Example 1 (Rapid Screening Technology for CRISPR-Cas9 Kinase Library): (1) Experimental steps: This experiment used the CRISPR-Cas9 kinase genome screening technology to identify host kinase targets dependent on Zika virus infection. The specific procedures were as follows: First, A549-Cas9 cells (ATCC CCL-185, stably expressing Cas9 protein after lentiviral infection) were screened at 5 × 10⁻⁶ cells per cell line. 5Cells were seeded at a density of 6-well plates and infected with lentivirus using the Addgene #101927 kinase sgRNA library (containing 2333 kinase genes) (MOI=0.3, infection enhancer polybrene 8 μg / mL). Forty-eight hours after infection, cells were screened for 7 days using medium containing 5 μg / mL puromycin (Solarbio #H8761) to obtain a stably transfected cell library (CRISPR-Cas9 kinase knockout library cells A549). Subsequently, the ZIKV SZ-WIV01 strain (preserved by the Molecular Epidemiology Laboratory of the Institute of Medical Biology, Chinese Academy of Medical Sciences, publicly available) was inoculated at MOI=1, with an uninfected group serving as a control. After 72 hours of culture until a significant cytopathic effect (CPE) was observed, genomic DNA was extracted using the CWBIO #CW2298M genomic DNA extraction kit and sent to Qingke Biotechnology Co., Ltd. for genome sequencing analysis.

[0028] (2) Experimental results: Analysis using the MAGeCK algorithm (FDR < 0.05, |log2FC| > 1) identified 20 kinase genes that were significantly absent in ZIKV-infected cells (see Table 1): Table 1

[0029] Among the top 20 significantly enriched candidates, three targets (dsRNA-activated protein kinase PRKRA (PMID: 33479180; PMID: 38793607), nuclear factor κB kinase subunit inhibitor β-IKBKB (PMID: 36788451), and early endosomal antigen 1 EEA1 (PMID: 38109552)) were consistent with CRISPR-Cas9 screening reported in other studies. Furthermore, given that cyclin-dependent kinase 2 (CDK2) is involved in the life cycle of SARS-CoV-2, cytomegalovirus, and hepatitis B virus (PMID: 34884662; PMID: 36575184; PMID: 35181784) and plays a crucial regulatory role, we focused on the CDK2 molecule to investigate its specific role in Zika virus infection. Overexpression of CDK2 in A549 / CaCo2 cells (ATCCCCL-185) (ATCC HTB-37) significantly promoted ZIKV nucleic acid replication and protein expression (see appendix). Figure 1 BE).

[0030] (3) Principle Analysis: This experiment successfully established a CRISPR-Cas9-based kinaseome screening system: ① The use of A549-Cas9 cells ensured high gene editing efficiency; ② Optimized lentiviral infection conditions (MOI=0.3) guaranteed single-copy sgRNA integration, avoiding gene dose effects; ③ Strict statistical criteria (FDR<0.05) ensured the reliability of the screening results. Several clinical-stage kinase inhibitors with existing human safety data (such as the CDK2 inhibitor SNS-032) target the key kinases identified in this experiment, greatly enhancing the translational potential of the research results. This screening system provides important target resources for the development of anti-ZIKV drugs targeting host factors.

[0031] Example 2 (In vitro anti-ZIKV experiment): (1) Experimental steps: This study systematically evaluated the anti-ZIKV activity of two clinical-stage CDK2 inhibitors (SNS-032 and Fadraciclib). First, a dose-response experiment was conducted: A549 cells (ATCC CCL-185) were injected with 1 × 10⁻⁶ cells. 4 Cells were seeded at a density of 0.1–10 μM in 96-well plates and pretreated for 4 hours with a gradient of inhibitor concentrations from 0.1–10 μM. After 1 hour of infection with ZIKV SZ-WIV01 strain (MOI=1), the medium was replaced with maintenance medium (DMEM + 2% FBS) containing the same concentration of inhibitor (Procell #PM150218) (Cell-box #AUS-01S-02). Cell viability was measured using a CCK-8 assay kit (Solarbio #CA1210) 48 hours later. 50 ) and qPCR detection of viral RNA levels (EC) 50 Based on EC 50As a result, a 2 μM concentration was selected for subsequent experiments: A549 and CaCo2 cells (ATCC HTB-37) were pretreated with the inhibitor for 4 hours, then infected with ZIKV (MOI=1) for 1 hour before the maintenance medium was replaced. Cells were collected 48 hours later for Western blot analysis (anti-ZIKV NS5 antibody 1:5000, GeneTex #GTX133327; GAPDH 1:5000, Proteintech #60004-1-Ig). To determine the dose-effect, inhibitor concentration gradients of 1 / 2 / 3 μM were set up. A549 cells were pretreated for 4 hours, then infected with ZIKV (MOI=1) for 1 hour before the maintenance medium was replaced. Cells were collected 48 hours later for Western blot analysis. To determine the action phase, three treatment modes were set up: ① pretreatment group (-4 to 0 hpi); ② co-treatment group (0-1 hpi); ③ post-treatment group (1-48 hpi), each group using a 2 μM concentration. All experiments were performed in triplicate.

[0032] (2) Experimental results: Dose-effect experiments showed that SNS-032 had an ECG effect on ZIKV. 50 The value was 1.714 ± 0.213 μM (CC). 50 =3.373±0.425 μM), with a selectivity index (SI) of 1.97 (see attached). Figure 2 A) Fadraciclib's EC 50 The value was 2.172 ± 0.187 μM (CC). 50 =4.520±0.362 μM), SI=2.08 (Appendix) Figure 2 B). Western blot analysis (with appendix) Figure 2 C) showed that 2 μM treatment reduced ZIKV NS5 protein expression in A549 / CaCo2 cells by 60-70% (SNS-032) and 40-50% (Fadraciclib) (n=3). Dose-dependent experiments showed (see attached). Figure 2 (D) The inhibitory effects of the two inhibitors, SNS-032 and Fadraciclib, on ZIKV are dose-dependent; the inhibitory effect increases with increasing drug concentration. (Time-series experiments are attached.) Figure 2 EG results showed that the pretreatment group had the strongest inhibitory effect (NS5 reduction of 50-60%), followed by the co-treatment group (30-40%), and the post-treatment group still had significant inhibition (30-40%).

[0033] (3) Principle Analysis: Experimental results reveal that CDK2 inhibitors inhibit ZIKV through multiple stages of action: ① Significant pretreatment effects suggest that they may affect the cellular state required for viral entry by regulating the host cell cycle (G1 / S phase arrest); ② The inhibitory effect in the co-treatment group indicates that it may directly interfere with the binding of the virus to the host receptor or the membrane fusion process; ③ Sustained late-stage inhibition suggests that it may also exert its effects by inhibiting viral RNA replication or protein synthesis. The similar inhibition patterns of the two inhibitors (pretreatment > co-treatment > post-treatment) confirm that CDK2 plays a key role in multiple stages of the ZIKV life cycle. The inherent favorable pharmacokinetic properties of the clinical-stage compounds (blood-brain barrier penetration, etc.) make them particularly suitable for the treatment of ZIKV infection.

[0034] Example 3 (Experiment on in vivo resistance to ZIKV infection): (1) Experimental steps: This experiment used 5-week-old A129 mice (type I interferon receptor deficient) to establish a ZIKV infection model. The specific procedures were as follows: mice were inoculated via intraperitoneal injection (ip) of 10... 5 PFU ZIKV virus strain SZ-WIV01 (amplified and preserved in the laboratory) was randomly assigned to a treatment group and a control group (n=5 / group) after inoculation. The treatment group received daily intraperitoneal injections of 10 mg / kg SNS-032 (MCE #HY-10008), 15 mg / kg Fadraciclib (MCE #HY-101212), and 0.5 mg / kg Temoporfin (positive control, MCE #HY-16488) for days 1-5 post-infection, while the control group received an equal volume of DMSO. Body weight changes were monitored and recorded daily. From days 2-9 post-infection, 50 μL of whole blood was collected via the tail vein, and RNA was extracted by TRIzol (Invitrogen #15596026CN). Mice were euthanized on day 9 post-infection, and blood, liver, and brain tissue were collected. One tissue sample was preserved in TRIzol for qPCR detection (primer sequences: ZIKV-NS5-F: 5'-TAAACGGGGTTGTCAGGCTC-3', R: 5'-ACCTGACGAGTGCCTTCTTG-3'); another sample was used to extract protein using RIPA (Solarbio #R0010) lysis buffer for Western blot analysis (anti-ZIKV NS5 antibody 1:5000, GeneTex #GTX133327).

[0035] (2) Experimental results: Experimental results show (attached) Figure 3The treated mice showed significant antiviral effects: ① Regarding viral replication, the level of ZIKV RNA in the blood of the treated group was lower than that of the control group (see appendix). Figure 3 C), viral RNA was significantly reduced in brain tissue and liver (see appendix) Figure 3 DE); ② Western blot showed a decrease in NS5 protein expression (see appendix). Figure 3 F); ③ Regarding clinical indicators, the weight loss in the treatment group was less severe than that in the control group (see appendix). Figure 3 B), in the treatment group, the levels of IL-6 and TNF-α in brain tissue and liver were downregulated (see appendix). Figure 3 G).

[0036] (3) Principle Analysis: The results of this experiment confirm that CDK2 inhibitors effectively inhibit ZIKV infection in vivo: ① The A129 mouse model, lacking a type I interferon response, can simulate the severe infection process in immunocompromised individuals, and the experimental results have clinical reference value; ② Viral RNA detection showed that the inhibitors significantly inhibited viral replication in the blood and target organs, suggesting that they may act by blocking viral RNA replication or assembly; ③ Changes in body weight and improved levels of inflammatory factors indicate that the inhibitors not only have direct antiviral effects but also alleviate virus-induced immunopathological damage; ④ Consistent viral inhibition in brain tissue and liver indicates that the compound has good tissue distribution characteristics. These findings provide important preclinical evidence for the treatment of ZIKV infection with CDK2 inhibitors.

[0037] Example 4 (In vitro study of its activity against dengue virus infection): (1) Experimental steps: This experiment used the A549 human lung cancer cell line (ATCC CCL-185) to evaluate the inhibitory effect of CDK2 inhibitors on dengue virus. The specific procedure was as follows: cells were cultured in DMEM medium containing 10% fetal bovine serum until 80% confluence, and then seeded into 12-well plates (2 × 10⁻⁶ cells / well). 5 Cells / well). Experimental groups were pretreated for 4 hours with 0, 1, 2, and 3 μM SNS-032 or Fadraciclib, respectively, followed by inoculation with DENV-1, DENV-2, DENV-3, and DENV-4 (the four serotypes of DENV were isolated, cultured, and preserved in the laboratory and are publicly available). The multiple of infection (MOI) was set to 1. One hour after virus adsorption, the medium was replaced with maintenance medium (DMEM + 2% FBS) containing the same concentration of inhibitor, and cultured for another 48 hours. Cell lysates were collected, and the expression level of viral E / 4G2 protein was detected by Western blot.

[0038] (2) Experimental results: Western blot results (see attached) Figure 4 Both CDK2 inhibitors showed dose-dependent inhibitory effects against all four DENV serotypes. Within the 1-3 μM concentration range, the expression level of DENV NS3 protein was significantly lower in the SNS-032 treatment group compared to the control group, and the same was true in the Fadraciclib group. There was no significant difference in inhibitory effect among the four serotypes, indicating that the inhibitors have broad-spectrum antiviral activity. Cell morphology observation showed no significant cytotoxicity at a concentration of 3 μM, consistent with previous clinical data.

[0039] (3) Principle Analysis: Experimental results confirm that CDK2 inhibitors inhibit DENV replication in a dose-dependent manner. The mechanism of action may involve: 1) CDK2, as a host-dependent factor, participates in regulating the cell cycle process required for viral RNA replication; the inhibitor inhibits viral proliferation by blocking this process; 2) Flavivirous viruses (including DENV and ZIKV) all depend on similar host factors to complete their replication cycle, which explains the broad-spectrum inhibitory effect of the inhibitors on multiple serotypes; 3) The selection of inhibitors in the clinical stage ensures that the compounds have good safety and pharmacokinetic properties, laying the foundation for subsequent translational research. This finding provides experimental evidence for the development of broad-spectrum antiflavial drugs targeting host factors.

[0040] The various embodiments of this application have been described above. These descriptions are exemplary and not exhaustive, nor are they limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments. The terminology used herein is chosen to best explain the principles, practical application, or improvement of the technology in the market, or to enable others skilled in the art to understand the embodiments disclosed herein.

Claims

1. A method for rapidly screening kinase inhibitors against Zika virus infection, characterized in that, The method includes the following steps: (1) Construct a stable cell bank for transfection; (2) After the stable transfection cell bank in (1) is constructed, the cells are inoculated with ZIKV SZ-WIV01 strain. After the cytopathic effect appears, the sgRNA region is amplified by PCR and high-throughput sequencing analysis is performed. (3) Compare the differences in sgRNA abundance between the infected group and the control group, screen for sgRNAs that are significantly missing in the infected cells, and sort the candidate genes to obtain the kinase target against Zika virus infection; the kinase target is CDK2.

2. The method according to claim 1, characterized in that, The MOI of the ZIKV SZ-WIV01 strain is 1.

3. The method according to claim 1, characterized in that, The cell library is a CRISPR-Cas9 kinase knockout library of cells, A549.

4. The method according to claim 1, characterized in that, In (3), the MAGeCK algorithm is used to compare the difference in sgRNA abundance between the infected group and the control group.

5. The method according to claim 1, characterized in that, In (3), candidate genes are ranked based on statistical significance.

6. The application of a screened CDK2 inhibitor in the preparation of anti-ZIKV drugs, characterized in that, The application does not involve the process of preventing or treating diseases.

Images