Application of Akti-1 / 2 in preparation of medicine for resisting new coronavirus

Abstract

The invention provides an application of Akti-1 / 2 in preparation of an anti-new coronavirus drug. And the new coronavirus is SARS-CoV-2 (Sarcabose Region The chemical name of the Akti-1 / 2 is 1, 3-dihydro-1-[1-[4-(6-phenyl-1H-imidazo [4, 5-g] quinoxaline-7-yl) phenylmethyl]-4-piperidinyl]-2H-benzimidazole-2-ketone, in-vitro Vero cell infection model research shows that the Akti-1 / 2 can inhibit replication of an original strain of the new coronavirus (SARS-CoV-2), and the activity of resisting the new coronavirus is achieved. In a Vero cell infection model, the IC50 (half maximal inhibitory concentration) of the SARS-CoV-2 original strain is 0.039 [mu] M. Therefore, the Akti-1 / 2 can be used for research and development of anti-new coronavirus infection drugs, and has a good development prospect.

Description

Technical Field

[0001] The present invention belongs to the technical field of antiviral drugs, and specifically relates to the use of Akti-1 / 2 in the preparation of anti-new coronavirus drugs. Background Art

[0002] The novel coronavirus (SARS-CoV-2) is the pathogen that causes coronavirus disease 2019 (COVID-19). Currently, the novel coronavirus continues to mutate, with new variants potentially becoming more contagious and pathogenic, potentially negating pre-existing immunity from vaccination or previous infection, and developing resistance to existing antiviral drugs. Therefore, treatment and prevention measures for the novel coronavirus and its variants have been a research hotspot in recent years.

[0003] At present, the research and development strategies for anti-new coronavirus drugs mainly include the design and development of targeted drugs for key viral molecules (such as Mpro, RdRP, PLpro, etc.) and host factors (such as ACE2, TMPRSS2, etc.), as well as drug screening based on viral infection models. The former develops inhibitors such as small molecules, peptides, and aptamers through virtual screening or experimental screening based on molecular interactions, and develops neutralizing antibodies through antigen immune screening or antibody gene sequencing. The latter screens drugs or compounds that can inhibit viral replication by testing various existing drug and compound libraries (including known active compound libraries, natural product compound libraries, etc.). The present invention found that Akti-1 / 2 has the activity of inhibiting the replication of the new coronavirus by screening the kinase inhibitor library.

[0004] Akti-1 / 2 (AKT inhibitor VIII) is a cell-permeable quinoxaline compound that selectively and reversibly inhibits the activity of Akt1, Akt2, and Akt3 with IC50 values ​​of 58 nM, 210 nM, and 2119 nM, respectively. Akt / protein kinase B (PKB)-mediated signaling pathways play important roles in cell proliferation, apoptosis, metabolism, and other processes. Akti-1 / 2 has potential anti-tumor activity. In vitro studies have found that Akti-1 / 2 can significantly promote apoptosis induced by tumor necrosis factor (TRAIL) or chemotherapy drugs (camptothecin, Herceptin, doxorubicin, etc.) in tumor cell lines such as prostate cancer (LnCaP), breast cancer (MCF7), colorectal cancer (HT29) and ovarian cancer (A2780) (Lindsley, et al. Allosteric Akt (PKB) inhibitors: discovery and SAR of isozyme selective inhibitors. Bioorg. Med. Chem. Lett. (2005), 15 (3), 761-764.). In lung cancer cell lines (HCC827, NCI-H522, NCI-1651 and PC-9), Akti-1 / 2 combined with gefitinib can synergistically inhibit cell growth (Puglisi M, et al. AKT inhibition synergistically enhances growth-inhibitory effects of gefitinib and increases apoptosis in non-small cell lung cancer cell lines. Lung Cancer. (2014), 85 (2): 141-6.). However, the therapeutic effect of Akti-1 / 2 in novel coronavirus infection has not been studied. The results of the present invention found that Akti-1 / 2 can inhibit the replication of novel coronavirus, and the specific mechanism needs further study. Summary of the Invention

[0005] The object of the present invention is to provide the use of Akti-1 / 2 (AKT inhibitor VIII) in the preparation of anti-new coronavirus drugs, wherein the Akti-1 / 2 (AKT inhibitor VIII) includes its pharmaceutically acceptable salts or other derivatives, the new coronavirus is SARS-CoV-2, the chemical name of the Akti-1 / 2 is: 1,3-dihydro-1-[1-[4-(6-phenyl-1H-imidazo[4,5-g]quinoxaline-7-yl)phenylmethyl]-4-piperidinyl]-2H-benzimidazol-2-one, the molecular formula of Akti-1 / 2 is: C34H29N7O, and the structural formula is as follows:

[0006] The present invention reveals that Akti-1 / 2 has the effect of inhibiting the replication of the new coronavirus. Studies in an in vitro Vero cell infection model show that Akti-1 / 2 can inhibit the replication of the original strain of the new coronavirus (SARS-CoV-2) and has anti-new coronavirus activity. In the Vero cell infection model, the IC50 against the original strain of SARS-CoV-2 was 0.039 μM. Akti-1 / 2 can be used to develop drugs against new coronavirus infection and has good development prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0007] Figure 1 In the Vero cell infection model, Akti-1 / 2 can significantly inhibit the replication of the new coronavirus, with an IC50 of 0.039μM against the original strain of SARS-CoV-2. DETAILED DESCRIPTION

[0008] The technical solution of the present invention will be clearly and completely described below in conjunction with the accompanying drawings and embodiments. The embodiments of the present invention include but are not limited to the following conditions and can be modified within the scope acceptable in the art. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without making any creative work shall fall within the scope of protection of the present invention.

[0009] Example 1: Anti-COVID-19 Drug Screening Model

[0010] African green monkey kidney Vero cells were cultured in DMEM medium at 37°C in a 5% CO2 incubator. The day before drug screening, an appropriate number of Vero cells were seeded in 48-well plates so that the density reached more than 90% the next day. The small molecule kinase inhibitor library was purchased from a commercial company with an initial concentration of 10 mM. First, it was diluted to an initial concentration of 1 mM with DMEM medium, and then diluted to four concentrations in a 10-fold gradient. 5 μL of the diluted drug was aspirated and added to the Vero cells in the culture plate, with a medium content of 495 μL per well. Next, the new coronavirus infection was carried out in a biosafety level 3 laboratory. The original virus solution was diluted with DMEM medium in an appropriate proportion, and 10 μL of the diluted venom was added to the culture wells to make the final infection titer reach approximately 100 times TCID50. The final drug concentrations were 10, 1, 0.1, 0.01 and 0.001 μM, respectively. At the same time, a negative control without virus infection and a positive control with virus but no drug were set up, all with 3 replicates. Three days after viral infection, the cell culture supernatant was collected to detect viral amplification and evaluate the inhibitory effect of the drug on viral replication. At the same time, the cell pathological changes were observed under a microscope. If high concentrations of the drug were found to cause severe cell pathological changes, the drug concentration was considered ineffective.

[0011] Example 2: Evaluation of anti-COVID-19 effect

[0012] After the cultivation is completed, 200 μL of the culture supernatant was aspirated and viral nucleic acid was extracted using a magnetic bead nucleic acid extraction kit and a fully automatic nucleic acid extractor (Shanghai Zhijiang Biology). The new coronavirus nucleic acid detection kit (fluorescence PCR method) was used to detect the level of viral RNA: by combining one-step RT-PCR with TaqMan technology, specific primers and probes were designed on the ORF1a / b and N genes, respectively. The ORF1a / b gene probe was labeled with FAM fluorescein, the N gene probe was labeled with VIC fluorescein, and the internal standard probe was labeled with ROX fluorescein. After PCR denaturation, annealing, and extension, the light signals emitted by the probes labeled with different fluorescein after hydrolysis were collected by the PCR instrument, and the amplification curve was presented on the instrument to realize the detection of the new coronavirus ORF1a / b and N genes. The level of the virus is represented by the Ct value, and the 2 -ΔΔCt Methods The relative amount of viral nucleic acid in the drug-treated group at each concentration and the drug-free group was calculated to obtain the percentage of viral inhibition and evaluate the anti-new coronavirus activity of the drug. Figure 1 .

[0013] In summary, the present invention provides a new use of Akti-1 / 2 against the novel coronavirus. Akti-1 / 2 is a potential drug for treating novel coronavirus infection.

Claims

1. The use of Akti-1 / 2 in the preparation of anti-new coronavirus drugs, characterized in that: The Akti-1 / 2 includes pharmaceutically acceptable salts thereof. The chemical name of Akti-1 / 2 is: 1,3-dihydro-1-[1-[4-(6-phenyl-1H-imidazo[4,5-g]quinoxalin-7-yl)phenylmethyl]-4-piperidinyl]-2H-benzimidazol-2-one, the molecular formula is: C34H29N7O, and the structural formula is as follows:

2. The use according to claim 1, characterized in that The new coronavirus is SARS-CoV-2.

3. The use according to claim 1, characterized in that Akti-1 / 2 exhibits anti-COVID-19 activity by inhibiting the replication of the original strain of SARS-CoV-2.

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