Use of plx8394 in the manufacture of a medicament for combating zika virus

Abstract

The application provides application of PLX8394 in preparation of an anti-ZIKV drug, a structural formula of the PLX8394 is shown as formula 1, the application firstly finds that the PLX8394 can be used for treating infectious diseases caused by ZIKV, and experiments show that the PLX8394 has extremely strong antiviral activity on ZIKV, can significantly inhibit replication of ZIKV on host cells A549, improve cell survival rate, can inhibit the process of virus entering the host cells A549, and has extremely strong antiviral effect at a cell level, and has obvious anti-ZIKV effect.

Description

Technical Field

[0001] This invention patent relates to the field of pharmaceutical technology, and in particular to the application of PLX8394 in the treatment of ZIKV infection. Background Technology

[0002] Zika virus (ZIKV) is a member of the Flavivirus genus within the Flaviviridae family. It is the most common pathogen causing microcephaly and Guillain-Barré syndrome in newborns. Since its first report in 1947, ZIKV-related infectious diseases have experienced numerous outbreaks and epidemics worldwide. Currently, the prevention and treatment of viral diseases mainly rely on vaccines and drugs. However, there is no vaccine available to prevent ZIKV infection, and treatment options for ZIKV-related infectious diseases are quite limited. The main treatment methods are symptomatic treatment, supportive treatment, and broad-spectrum antiviral therapy; however, these methods exhibit significant individual variability and are difficult to implement universally. Therefore, the development of specific and effective anti-ZIKV drugs is imperative.

[0003] During ZIKV invasion of host cells, it hijacks cell signaling pathways and degrades specific host proteins to promote its own replication. Therefore, viral-supporting cell signaling pathways may be promising novel antiviral targets. The RAF / MEK / ERK signaling pathway belongs to the classic mitogen-activated protein kinase (MAPK) cascade pathway and plays an important role in cell growth and development. The RAF family includes A-RAF, B-RAF, and C-RAF, which are serine / threonine protein kinases that can form RAF homodimers or heterodimers, activating downstream MEK1 / 2 proteins through phosphorylation; MEK kinases activate downstream ERK1 / 2 through MEK phosphorylation.

[0004] Therefore, it is essential to develop an effective drug against the Zika virus. Summary of the Invention

[0005] PLX8394 is a novel RAF inhibitor that inhibits ERK signaling by specifically inhibiting B-RAF dimers, including B-RAF homodimers and B-RAF:C-RAF heterodimers, but it does not disrupt C-RAF homodimers or A-RAF-containing dimers. The difference in amino acid residues at the N-terminus of the RAF isoform kinase domain is the reason for this differential vulnerability. PLX8394 selectively inhibits ERK signaling in tumors driven by B-RAF dimer mutants, but retains RAF function in normal cells where C-RAF homodimers can drive signaling. Therefore, PLX8394 shows promising therapeutic effects against B-RAF mutation-driven metastatic melanoma and metastatic colorectal cancer. Currently, there are reports of PLX8394's inhibitory effect on enterovirus EV71, but its anti-ZIKV infection activity has not been reported.

[0006] The structure of PLX8394 is shown in Equation 1 below:

[0007]

[0008] The present invention aims to at least partially solve one of the technical problems existing in the prior art. Therefore, in a first aspect, the present invention provides the application of PLX8394 in the preparation of anti-Zika virus drugs. The structural formula of PLX8394 is shown in Formula 1.

[0009]

[0010] In a second aspect, the present invention provides the use of PLX8394 in the preparation of drugs for the prevention of Zika virus infection, wherein the structural formula of PLX8394 is shown in Formula 1.

[0011]

[0012] In one or more embodiments of the present invention, the Zika virus infection prevention drug is a Zika virus infection prevention drug.

[0013] In one or more embodiments of the present invention, the concentration of PLX8394 in the anti-Zika virus drug or the drug for preventing Zika virus infection is 0.01-10 μM.

[0014] Preferably, the concentration of PLX8394 in the anti-Zika virus drug or the drug for preventing Zika virus infection is 0.1-10 μM.

[0015] More preferably, the concentration of PLX8394 in the anti-Zika virus drug or the drug for preventing Zika virus infection is 1-10 μM. The inventors have experimentally discovered that when the concentration of PLX8394 is 1 μmol / L, a 50% inhibition rate against Zika virus can be achieved.

[0016] In one or more embodiments of the present invention, the application includes the combination of PLX8394 and ribavirin.

[0017] In one or more embodiments of the present invention, the anti-Zika virus drug or the drug for preventing Zika virus infection further includes pharmaceutically acceptable excipients and carriers.

[0018] In one or more embodiments of the present invention, the dosage form of the anti-Zika virus drug or the drug for preventing Zika virus infection is granules, tablets, pills, capsules or injections.

[0019] The inventors evaluated the inhibitory activity of PLX8394 against ZIKV using standard viral activity assays. Extensive biological experiments revealed that PLX8394 exhibits good inhibitory activity against ZIKV entry into cells. Therefore, PLX8394 demonstrates an inhibitory effect against ZIKV.

[0020] The beneficial effects of this invention are as follows:

[0021] 1. Application of PLX8394 in the preparation of anti-Zika virus drugs: This invention is the first to discover that PLX8394 can be used to treat infectious diseases caused by ZIKV. Experiments show that PLX8394 has extremely strong antiviral activity against ZIKV, can significantly inhibit the replication of ZIKV in host cells A549, improve cell survival rate, and inhibit the process of virus entering host cells A549. It exhibits extremely strong antiviral effects at the cellular level and has obvious anti-ZIKV effects.

[0022] 2. Application of PLX8394 in the preparation of drugs for the prevention of Zika virus infection. This invention is the first to discover that PLX8394 can more effectively inhibit the virus when administered before or during Zika virus invasion. Attached Figure Description

[0023] Figure 1 This is a graph showing the A549 cell cytotoxicity test results of PLX8394 against ZIKV in Example 1 of this invention;

[0024] Figure 2 This is a graph showing the safety and efficacy results of PLX8394 as an antiviral drug in Example 2 of the present invention;

[0025] Figure 3This is a schematic diagram of the immunofluorescence of PLX8394 and ribavirin inhibiting ZIKV virus at concentrations of 0.1 μM and 1 μM.

[0026] Figure 4 Figure 1 shows the inhibitory effect of different concentrations of PLX8394 on the expression level of ZIKV viral proteins.

[0027] Figure 5 The graph shows the inhibitory effect of different concentrations of PLX8394 on ZIKV mRNA levels.

[0028] Figure 6 Figure 1 shows the inhibitory effect of different concentrations of ribavirin on the expression level of ZIKV viral proteins.

[0029] Figure 7 A schematic diagram of the timeline processed by PLX8394;

[0030] Figure 8 To investigate the effect of PLX8394 on the expression level of ZIKV viral proteins at different stages of ZIKV infection;

[0031] Figure 9 The inhibitory effect of PLX8394 on the adsorption and cell invasion stages of ZIKV;

[0032] Figure 10 The inhibitory effect of PLX8394 on ZIKV before and after cell invasion. Detailed Implementation

[0033] The present invention will be further described below with reference to specific embodiments and accompanying drawings. However, the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified, specific conditions in the following embodiments are performed under conventional conditions or conditions recommended by the manufacturer. Unless otherwise specified, the methods used are conventional methods known in the art, and the consumables and reagents used are commercially available. Unless otherwise stated, the technical and scientific terms used herein have the same meaning as those familiar to those skilled in the art. Furthermore, any methods or materials similar to or equivalent to those described herein may also be applied to the present invention.

[0034] In the following embodiments, this invention combines viral RNA level analysis, titer determination, and Alamar Blue cell viability assay to study the anti-ZIKV activity of PLX8394. Data analysis in these embodiments was performed using GraphPadPrism 9.0 software. It is understood that further optimization using PLX8394 as a lead compound for the preparation of drugs to treat ZIKV infectious diseases also falls within the scope of this invention.

[0035] Example 1: Cytotoxicity detection of PLX8394

[0036] The cytotoxicity of PLX8394 was assessed in A549 cells. A549 cells were cultured at a rate of 1 × 10⁻⁶ cells / year. 4 Cells were evenly seeded per well in 96-well plates and cultured at 37°C in a 5% CO2 incubator for 12-16 hours. The cell culture medium was then discarded, and culture medium containing different concentrations of PLX8394 and 2% FBS was added for further culture. Each group had six replicates, with an equal volume of 2% FBS culture medium used as a control. After 48 hours of incubation, the cells were stained with Alamar Blue and incubated at 37°C for 2 hours. Fluorescence values ​​were measured using a multi-mode microplate reader (fluorescence detection: excitation at 560 nm, emission wavelength at 590 nm) to analyze cell viability.

[0037] Results Analysis: The test results are as follows: Figure 1 As shown, GraphPad Prism 9.0 software calculated the median cyctoxic concentration (CC50) of the drug for cells, and the CC50 of PLX8394 was 75.86 μmol / L. In subsequent implementation cases, the maximum concentration of PLX8394 used was 10 μM, which is within the safe and non-toxic range.

[0038] Example 2: Safety and efficacy analysis of PLX8394 as an antiviral drug

[0039] A549 cells were uniformly seeded in 6-well cell culture plates and cultured at 37°C in a 5% CO2 incubator for 12-16 h. After infection with ZIKV at MOI=2 for 1 h, cells were treated with cell maintenance medium containing different concentrations of PLX8394. The negative control was treated with the same volume of 2% FBS culture medium. After 24 h of treatment, the effect of inhibiting viral proliferation was detected by RT-qPCR.

[0040] The inhibition rate of different concentrations of PLX8394 against ZIKV was calculated based on the viral copy number obtained by the absolute quantification method. Nonlinear regression analysis of the drug's inhibition rate against the virus was performed using GraphPad Prism 9.0 to calculate the half-maximal inhibitory concentration (IC50). The IC50 of PLX8394 was 1 μM. The selectivity index (SI) is a reference index used to determine the safety range of drug efficacy. Its value in in vitro experiments is equal to the ratio of CC50 / EC50. A selectivity index greater than 1 is considered to indicate that the drug is effective, and a higher index value indicates greater safety.

[0041] Results analysis: Based on the previous cytotoxicity test results (CC50 = 75.86 μmol / L), the selectivity index of PLX8394 can be calculated to be 75.86. This result further demonstrates that PLX8394 has good safety and efficacy in inhibiting ZIKV.

[0042] Example 3: Detection of antiviral activity of different concentrations of PLX8394 against ZIKV

[0043] A549 cells were seeded in 6-well cell culture plates and cultured at 37°C in a 5% CO2 incubator for 12–16 h. After infection with ZIKV (MOI = 2) for 1 h, cells were treated with 0.1 μM and 1 μM PLX8394 cell maintenance medium. A negative control was added with the same volume of 2% FBS culture medium. After 24 h of treatment, viral infection was detected using immunofluorescence. Subsequently, the same experiments were performed with different concentrations of PLX8394 (0.01 μM, 0.1 μM, 1 μM, and 10 μM). Viral protein expression levels were detected by Western blotting, and the inhibitory effect of PLX8394 on viral proliferation was detected by RT-qPCR. Ribavirin was used as a positive control to detect viral protein expression levels.

[0044] Results Analysis: The test results are as follows: Figure 3 As shown, Figure 3 This is a schematic diagram of the immunofluorescence of PLX8394 inhibiting ZIKV virus at concentrations of 0.1 μM and 1 μM. Figure 4 Inhibitory effect of different concentrations of PLX8394 on ZIKV viral protein expression levels; Figure 5 The inhibitory effect of different concentrations of PLX8394 on ZIKV mRNA levels; Figure 6 This study investigated the inhibitory effects of different concentrations of the positive control (Ribavirin) on ZIKV viral protein expression levels. Under different concentrations, PLX8394 inhibited ZIKV proliferation, with the inhibitory effect becoming more pronounced with increasing PLX8394 concentration. Furthermore, Figure 4 and Figure 6 The comparison shows that, at the same concentration, ribavirin's inhibitory effect on the virus is lower than that of PLX8394.

[0045] Example 4: Detection of antiviral activity of PLX8394 at different stages of ZIKV-treated cells

[0046] A549 cells were seeded in 6-well cell culture plates and cultured at 37°C in a 5% CO2 incubator for 12–16 h. The cells were then infected with ZIKV at an MOI of 2. At different stages of ZIKV treatment, A549 cells were treated with cell maintenance medium containing 1 μM PLX8394. A negative control was prepared with the same volume of 2% FBS. After 24 h of treatment, the inhibitory effect of PLX8394 on viral proliferation was detected by RT-qPCR, and the expression levels of viral proteins were detected by immunoblotting.

[0047] Results analysis: The antiviral activity of PLX8394 at different stages of ZIKV-treated cells was detected as follows: Figure 7-10 As shown, where, Figure 7 A schematic diagram of the timeline processed by PLX8394. Figure 8 To investigate the effect of PLX8394 on the expression levels of ZIKV viral proteins at different stages of ZIKV infection. Figure 9 This demonstrates the inhibitory effect of PLX8394 on the adsorption and cell invasion phases of ZIKV. Figure 10 This study investigated the inhibitory effects of PLX8394 on ZIKV before and after its invasion of cells. The results showed that PLX8394 significantly inhibited ZIKV during the invasion phase and also exhibited some inhibitory effect before invasion.

[0048] In summary, PLX8394 can significantly inhibit ZIKV invasion activity, significantly suppress the expression level of viral proteins after A549 infection with ZIKV, inhibit viral RNA replication, and enhance cell survival rate. It has the potential to be further developed into a clinically effective drug against ZIKV infection.

[0049] This invention, through the above embodiments, confirms the role of PLX8394 in anti-ZIKV. PLX8394 can inhibit the expression level of viral proteins and RNA replication of ZIKV in host cells A549, enhancing cell viability; it has the potential to be developed into an effective drug for treating ZIKV infection. This invention further provides the application of PLX8394 in the preparation of anti-ZIKV infection drugs. This application refers to the addition of pharmaceutically acceptable excipients and carriers to PLX8394 for the preparation of anti-ZIKV formulations. The excipients include at least one of fillers, disintegrants, binders, excipients, diluents, lubricants, sweeteners, or colorants, with different excipients selected according to the needs of the drug dosage form. The formulation is a granule, tablet, pill, capsule, injection, or dispersant.

[0050] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention, all of which should be included within the protection scope of the present invention.

Claims

1. The application of PLX8394 in the preparation of drugs for the prevention of Zika virus infection, characterized in that, The structural formula of PLX8394 is shown in Equation 1. 1。 2. The application of PLX8394 according to claim 1 in the preparation of drugs for preventing Zika virus infection, characterized in that, The drug mentioned is for preventing severe Zika virus infection.

3. The application according to claim 1, characterized in that, The concentration of PLX8394 in the drug for preventing Zika virus infection is 0.01-10 μM.

4. The application according to claim 1, characterized in that, The concentration of PLX8394 in the drug for preventing Zika virus infection is 0.1-10 μM.

5. The application according to claim 1, characterized in that, This includes the combined use of PLX8394 and ribavirin.

6. The application according to claim 1, characterized in that, The drug for preventing Zika virus infection also includes pharmaceutically acceptable excipients.

7. The application according to claim 1, characterized in that, The dosage forms of the drugs for preventing Zika virus infection are granules, tablets, pills, capsules, or injections.

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