Application of aminoimidazole compound HY-W051988 and derivative thereof in resisting RNA (Ribonucleic Acid) virus

Abstract

The invention discloses an application of an aminoimidazole compound HY-W051988 and a derivative thereof in resisting RNA (Ribonucleic Acid) viruses, and relates to the technical field of biological medicines. The chemical formula of the amino imidazole compound HY-W051988 provided by the invention is C20H17N3O4, and the molecular weight of the amino imidazole compound HY-W051988 is 362.368. A large number of experimental studies prove that the HY-W051988 has a relatively good anti-RNA virus effect at a low concentration (400 nM), has a remarkable inhibition effect on various RNA viruses, and is relatively good in broad spectrum; the toxicity is small, and the normal growth of host cells is not influenced.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to application of aminoimidazole compound HY-W051988 and derivatives thereof in resisting RNA viruses. Background Art

[0002] Arl8 (ADP-ribosylation factor-like protein 8) is a member of the Arf-like (Arl) family of proteins and a small GTPase belonging to the Ras superfamily. It can act as a molecular switch by alternating between a GDP-bound inactive conformation and a GTP-bound active conformation. This process is well regulated by related GTPase-activating proteins (GAPs) and guanine nucleotide exchange factors (GEFs).

[0003] In mammals, there are two Arl8 homologs, Arl8a and Arl8b, which are highly similar to each other and are mostly located on the surface of lysosomes. Their main functions are lipid turnover and mediating lysosomal-regulated cargo transport (lysosomal transport). Once the GTP-bound active form of Arl8b is targeted to the lysosomal membrane, it can then mediate the recruitment of related effector proteins to the lysosomal membrane, including SKIP, HOPS complex, PLEKHM1, RUFY1, RUFY3, and RUFY4. This in turn promotes downstream lysosome-related cellular events, such as cellular lysosomal trafficking and positioning and fusion between lysosomes and autophagosomes. As an important host factor, Arl8b can help clear engulfed microorganisms in macrophages. However, when the host's cellular machinery is exploited by pathogenic microorganisms, it may help them move to the periphery of the cell and infect neighboring cells. This phenomenon is reflected in both animal and plant cells.

[0004] RNA viruses are viruses whose genetic material is RNA. They are divided into single-stranded and double-stranded viruses. They replicate in two ways: self-replication and reverse transcription. They have the characteristics of rapid mutation and difficulty in vaccine development. Common RNA viruses include HIV, influenza virus, hepatitis C virus, coronavirus, polio virus, etc. At the cellular molecular level, designing specific target protein inhibitors for treatment of already identified antiviral proteins can achieve the effect of specifically killing viruses in the host. However, no research results have been found so far that use Arl8 as a target protein, prepare corresponding antiviral preparations based on its mechanism of action, and conduct antiviral treatment. Summary of the invention

[0005] The present invention provides an application of an aminoimidazole compound HY-W051988 and its derivatives in anti-RNA virus. The present invention uses the small GTPase Arl8b of the host cell of RNA virus as a specific target, screens and obtains an aminoimidazole compound HY-W051988 or its derivatives, and through antiviral detection, it is found that this type of small molecule compound has a significant inhibitory effect on RNA viruses such as foot-and-mouth disease virus, enterovirus and vesicular stomatitis virus, and has no obvious toxic effect on the host cells corresponding to these RNA viruses. Therefore, the HY-W051988 provided by the present invention has good anti-RNA virus activity and good targeting specificity for RNA viruses, and can be used as an antiviral drug for research and development. It is specifically achieved by the following technologies.

[0006] In the first aspect of the present invention, an aminoimidazole compound HY-W051988 or a derivative thereof is provided, wherein the aminoimidazole compound HY-W051988 is used to prepare an anti-RNA virus preparation; the chemical molecular formula of the aminoimidazole compound HY-W051988 is C 20 H 17 N 3 O 4 , the relative molecular weight is 362.368, and the chemical structure is:

[0007] .

[0008] Furthermore, the aminoimidazole compound HY-W051988 or its derivatives target and inhibit the expression of Arl8b protein in host cells of RNA viruses.

[0009] Furthermore, the aminoimidazole compound HY-W051988 or its derivatives target and inhibit the lipophagy of the host cells of RNA viruses.

[0010] The present invention verifies the low toxicity of HY-W051988 to RNA virus host cells through CCK-8 experiments. Through qRT-PCR experiments, Western Blot experiments and TCID50 experiments, it is verified that the inhibitor HY-W051988 can inhibit FMDV replication. Through qRT-PCR experiments, the target of the inhibitor HY-W051988 is verified. The broad spectrum of inhibitor HY-W051988 in inhibiting viral replication is verified for enterovirus and vesicular stomatitis virus. Through lipophage experiments, the inhibitor HY-W051988 is verified to inhibit lipophage of infected cells. Through qRT-PCR experiments, the inhibitor HY-W051988 is verified to inhibit the broad spectrum of viral release in foot-and-mouth disease virus, enterovirus and vesicular stomatitis virus.

[0011] The second aspect of the present invention provides a use of any one of the above aminoimidazole compounds HY-W051988 or its derivatives in the preparation of anti-RNA virus preparations.

[0012] Furthermore, the RNA virus is foot-and-mouth disease virus, enterovirus, or vesicular stomatitis virus.

[0013] The third aspect of the present invention provides an anti-RNA virus preparation comprising any one of the above-mentioned aminoimidazole compounds HY-W051988 or a derivative thereof.

[0014] Furthermore, it also includes one or more medically or pharmaceutically acceptable carriers, excipients, and diluents.

[0015] Furthermore, the anti-RNA virus preparation is an anti-RNA virus drug, and the dosage form of the anti-RNA virus drug includes a solid oral preparation, a liquid oral preparation or an injection.

[0016] Furthermore, the dosage forms of the anti-RNA virus preparation include tablets, dispersible tablets, enteric-coated tablets, chewable tablets, orodisintegrating tablets, capsules, sugar-coated tablets, granules, dry powders, oral solutions, small water injections for injection, freeze-dried powder injections for injection, large infusions and small infusions.

[0017] Compared with the prior art, the present invention is beneficial in that:

[0018] 1. The aminoimidazole compound HY-W051988 or its derivatives provided by the present invention have obvious inhibitory effects on a variety of RNA viruses at low concentrations and have good broad-spectrum properties.

[0019] 2. The aminoimidazole compound HY-W051988 or its derivatives provided by the present invention has low toxicity to host cells of RNA viruses and does not affect the normal growth of host cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a real product photo of aminoimidazole compound HY-W051988.

[0021] Figure 2 The results show the toxicity of different concentrations of HY-W051988 on BHK-21 cells.

[0022] Figure 3 This is the effect of HY-W051988 treatment on the expression level of FMDV 3D in cells.

[0023] Figure 4 The figure shows the effect of HY-W051988 treatment at a final concentration of 400 nM on the level of FMDV 3D protein.

[0024] Figure 5 The figure shows the effect of treating cells with HY-W051988 at a final concentration of 1 μM on the viral titer.

[0025] Figure 6 The expression levels of Arl8b and FMDV 3D in cells after treatment with HY-W051988 and Arl8b overexpression.

[0026] Figure 7 The replication of EV71 was inhibited after treatment with HY-W051988.

[0027] Figure 8 The replication of VSV was inhibited after HY-W051988 treatment.

[0028] Fig. 9 Figure XX shows that HY-W051988 inhibits cell lipophagy.

[0029] Fig.10 HY-W051988 inhibits cell lipophagy.

[0030] Fig.11 HY-W051988 inhibits RNA viruses: FMDV, EV71 and VSV release. DETAILED DESCRIPTION

[0031] The technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0032] In some embodiments of the present invention, the chemical formula of the aminoimidazole compound HY-W051988 is C 20 H 17 N 3 O 4 , the relative molecular weight is 362.368, and the chemical structure is as follows:

[0033] .

[0034] The aminoimidazole compound HY-W051988 is prepared and provided by MCE (MedChemExpress); Figure 1 As shown, trade name: 4-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-1-methyl-1H-imidazole-2-car

[0035] -boxylic acid, specification model 1 mg; Cat.#: HY-W051988; Lot#: 222034; Storage conditions: Store at room temperature.

[0036] In the following specific implementation case, foot-and-mouth disease virus (FMDV O type, Akesu / 58 / 2002, provided by Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences), enterovirus 71 (EV71, provided by China Center for Type Culture Collection) and vesicular stomatitis virus (VSV, provided by China Center for Type Culture Collection) were selected as representatives of RNA viruses; Syrian hamster kidney cells BHK-21 (provided by China Center for Type Culture Collection) and African green monkey kidney cells Vero (provided by China Center for Type Culture Collection) were selected as host cell objects of RNA viruses, and detection and verification were performed respectively.

[0037] Example 1: Verification of HY-W051988 toxicity to host cells

[0038] This example uses CCK-8 test to detect the toxicity of HY-W051988 to host cells BHK-21. The specific test method is as follows:

[0039] (1) BHK-21 cells were cultured at 5×10 4 The cells were inoculated at a density of 100 cells per well in a 96-well plate, and 100 μL of cell suspension was inoculated in each well. When the cell confluence reached about 70-80%, the original culture medium in the well was aspirated and discarded, and fresh MEM culture medium containing aminoimidazole compound HY-W051988 (concentrations were 100 nM, 200 nM, 400 nM, 1 μM, 5 μM, and 10 μM, respectively) was added. Eight replicates were set for each concentration; DMSO was used as a blank control.

[0040] (2) At 37°C with 5% CO 2 Incubate in the incubator for 48 h, then discard the original culture medium in the wells, replace it with 100 μL of fresh culture medium containing 10% CCK-8 solution and incubate for 2 h.

[0041] (3) Use Labserv K3 microplate reader to measure the absorbance of each well at 450 nm. Calculate the cell survival rate based on the absorbance of each well. The calculation method of survival rate (%) is: survival rate = (OD 450 Sample / OD 450 Blank control) × 100%.

[0042] like Figure 2 As shown, it can be seen that the cell survival rate is high when different concentrations of HY-W051988 are added, indicating that different concentrations of HY-W051988 do not affect the normal growth of host cells.

[0043] Example 2: Verification of the inhibitory effect of HY-W051988 on FMDV replication

[0044] In this example, qRT-PCR experiments, Western Blot experiments and TCID 50 Experiment to verify the inhibitory effect of HY-W051988 on FMDV replication.

[0045] 1. Test of FMDV 3D expression level and FMDV 3D protein level by HY-W051988

[0046] (1) BHK-21 cells were seeded into 24-well plates. After spreading, the plates were gently shaken to ensure that the cells were evenly distributed. The plates were then placed in a 37°C incubator containing 5% CO. 2 incubator.

[0047] (2) After the cells adhered to the wall, they were infected with FMDV. 200 μL of virus solution was added to each well and the culture medium was incubated at 37 °C with 5% CO. 2 The cells were placed in an incubator for 1 h, then the medium was replaced with 2% MEM medium for continued culture, and compound HY-W051988 (final concentration 200 nM and 400 nM) was added, respectively.

[0048] (3) When the cells show CPE (Cytopathic Effect), collect samples and perform qRT-PCR and Western Blot detection.

[0049] The results are as follows Figure 3 and 4 As shown, from Figure 3 It can be seen that HY-W051988 at 200 nM and 400 nM concentrations can significantly inhibit FMDV at the RNA level compared to the control group (DMSO). Figure 4 It can be seen that under the same infection conditions, the expression of FMDV's characteristic protein 3D was significantly reduced after drug treatment. This shows that HY-W051988 can significantly inhibit FMDV at the RNA level and protein level at a concentration of 400 nM.

[0050] 2. Effect of HY-W051988 on virus titer

[0051] (1) Inoculate BHK-21 cells into a 96-well plate and gently shake the plate to ensure even distribution of the cells.

[0052] (2) After the cells adhered to the wall, dilute them with a gradient of 10 -1 Up to 10 -8Each well was infected with FMDV, and 100 μL of virus solution was added to each well. The cells were incubated at 37 °C with 5% CO 2 Incubator for 1 h.

[0053] (3) Change to 2% MEM medium and continue culturing. Add compound HY-W051988 (final concentration 1 μM) and detect TCID 50 .

[0054] The results are as follows Figure 5 As shown, after adding HY-W051988, TCID 50 This indicates that compound HY-W051988 can significantly inhibit viral replication and reduce viral titer.

[0055] Example 3: Verification of the mechanism of action of HY-W051988 in inhibiting FMDV

[0056] This example is used to verify the mechanism of action of HY-W051988 in inhibiting FMDV, and the specific method is as follows:

[0057] (1) BHK-21 cells were seeded into 24-well plates. After spreading, the plates were gently shaken to ensure that the cells were evenly distributed. The plates were then placed in a 37°C incubator containing 5% CO. 2 Incubate in an incubator for 24 h;

[0058] (2) The incubated BHK-21 cells were divided into two groups. The control group was transfected with an empty vector, and the experimental group was transfected with an Arl8b overexpression plasmid tagged with Myc. The cells were cultured for 24 h respectively.

[0059] (3) Infect with FMDV. Add 200 μL of virus solution to each well and incubate at 37°C with 5% CO 2 The cells were incubated in an incubator for 1 h, then the medium was changed to 2% MEM medium for continued culture, and compound HY-W051988 (final concentration 400 nM) was added.

[0060] (4) When the cells show CPE, collect samples and perform qRT-PCR detection.

[0061] The results are as follows Figure 6 As shown, it can be seen that the viral RNA level increased significantly after overexpression treatment, and under the same treatment conditions, the viral RNA level of the group with added compound HY-W051988 was lower than that of the group without addition. This indicates that Arl8b overexpression will promote viral replication, but the addition of compound HY-W051988 can inhibit this process.

[0062] Example 4: Verification of the broad spectrum of HY-W051988 inhibition of Arl8b in regulating viral replication

[0063] In order to verify the broad spectrum of the aminoimidazole compound HY-W051988 in regulating viral replication by inhibiting Arl8b, the aminoimidazole compound HY-W051988 was tested in RNA viruses (enterovirus 71 (EV71), vesicular stomatitis virus (VSV)) to verify the regulation of viral nucleic acid levels. The specific method is as follows:

[0064] (1) Inoculate the host Vero cells corresponding to the two RNA viruses into a 24-well plate, gently shake the plate to evenly distribute the cells, and place it in a 37°C incubator with 5% CO. 2 incubator.

[0065] (2) After cells adhere, use TCID 50 =10 -4 The viruses were used to infect the corresponding host cells, and 200 μL of virus solution was added to each well. The cells were incubated at 37 °C with 5% CO 2 Incubator for 1 h.

[0066] (3) Change to 2% MEM medium and continue culturing, and add HY-W051988 (final concentration 400 nM).

[0067] (4) When the cells show CPE, collect samples and perform qRT-PCR detection.

[0068] The results are as follows Figure 7 , Figure 8 As shown, it can be seen that after drug treatment, the viral RNA levels of EV71 and VSV decreased significantly. This shows that the aminoimidazole compound HY-W051988 has different degrees of inhibition on the nucleic acid levels of EV71 and VSV, indicating that the antiviral effect of HY-W051988 is broad-spectrum.

[0069] Example 5: Verification of HY-W051988's inhibition of lipophagy in host cells infected with RNA viruses

[0070] This example uses a lipophagy experiment to verify the inhibitor HY-W051988's inhibition of lipophagy in host cells infected by RNA viruses. The specific method is as follows:

[0071] (1) BHK-21 cells were seeded into 24-well plates. After spreading, the plates were gently shaken to ensure that the cells were evenly distributed. The plates were then placed in a 37°C incubator containing 5% CO. 2 incubator.

[0072] (2) After the cells adhere to the wall, use lentiviral transfection to add a green fluorescent tag to the PLIN2 protein and a red fluorescent tag to LC3 (autophagy marker - microtubule-associated protein 1 light chain 3).

[0073] (3) 48 h after transfection, infect with FMDV, add 200 μL of virus solution to each well, and incubate at 37 °C with 5% CO 2 Incubator for 1 h;

[0074] (4) Change to 2% MEM medium and continue culturing, and add compound HY-W051988 (final concentration 400 nM).

[0075] (5) When the cells show CPE, prepare sections and observe under an electron microscope.

[0076] The results are as follows Fig. 9 and 10 As shown in the figure, it can be seen that after virus infection, both red and green fluorescence increased, and then decreased significantly after adding drugs. Moreover, the two fluorescences highly overlapped. This indicates that HY-W051988 has a significant inhibitory effect on lipophagy in FMDV.

[0077] Example 6: Effect of HY-W051988 on virus release and verification of its broad spectrum

[0078] This example is used to verify the effect of HY-W051988 on virus release and its broad spectrum. The specific method is as follows:

[0079] (1) BHK-21 cells and Vero cells were seeded into 24-well plates. After spreading, the plates were gently shaken to ensure that the cells were evenly distributed. The plates were then placed in a 37°C incubator containing 5% CO. 2 incubator.

[0080] (2) After the cells adhered, they were infected with FMDV, EV71, and VSV, and the compound HY-W051988 (final concentration 400 nM) was added to each cell.

[0081] (3) After the cells show CPE, extract the extracellular RNA and perform qRT-PCR detection.

[0082] The results are as follows Fig.11 As shown, from Fig.11 It can be seen that the extracellular viral RNA levels of FMDV, EV71 and VSV decreased after adding the drug. This shows that HY-W051988 can inhibit the release of RNA viruses, including +RNA viruses (FMDV and EV71) and -RNA viruses (VSV), with a broad spectrum.

[0083] The above specific embodiments describe the implementation of the present invention in detail, but the present invention is not limited to the specific details in the above embodiments. Within the scope of the claims and technical concept of the present invention, the technical solution of the present invention can be modified and changed in many simple ways, and these simple modifications all belong to the protection scope of the present invention.

Claims

1. An aminoimidazole compound HY-W051988 or a derivative thereof, characterized in that: The aminoimidazole compound HY-W051988 is used to prepare an anti-RNA virus preparation; the chemical structure of the aminoimidazole compound HY-W051988 is: 。 2. The aminoimidazole compound HY-W051988 or its derivative according to claim 1, characterized in that: The aminoimidazole compound HY-W051988 or its derivatives target and inhibit the expression of Arl8b protein in host cells of RNA viruses.

3. The aminoimidazole compound HY-W051988 or its derivative according to claim 1, characterized in that: The aminoimidazole compound HY-W051988 or a derivative thereof targets and inhibits the lipophagy of the host cell of the RNA virus.

4. Use of the aminoimidazole compound HY-W051988 or its derivatives according to any one of claims 1 to 3 in the preparation of anti-RNA virus preparations.

5. The use of the aminoimidazole compound HY-W051988 or its derivatives according to claim 4 in the preparation of anti-RNA virus preparations, characterized in that: The RNA virus is foot-and-mouth disease virus, enterovirus, and vesicular stomatitis virus.

6. An anti-RNA virus preparation, characterized in that: The invention comprises the aminoimidazole compound HY-W051988 or a derivative thereof according to any one of claims 1 to 3.

7. The anti-RNA virus preparation according to claim 6, characterized in that It also includes one or more medically or pharmaceutically acceptable carriers, excipients, and diluents.

8. The anti-RNA virus preparation according to claim 6, characterized in that The anti-RNA virus preparation is an anti-RNA virus drug, and the dosage form of the anti-RNA virus drug includes a solid oral preparation, a liquid oral preparation or an injection.

9. The anti-RNA virus preparation according to claim 8, characterized in that The dosage forms of the anti-RNA virus preparation include tablets, dispersible tablets, enteric-coated tablets, chewable tablets, orally disintegrating tablets, capsules, sugar-coated tablets, granules, dry powders, oral solutions, small water injections for injection, freeze-dried powder injections for injection, large infusions and small infusions.

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