Application of CC-115 in preparation of medicine for resisting new coronavirus

Abstract

The invention provides an application of CC-115 in preparation of an anti-new coronavirus drug. The chemical name of the CC-115 is 1-ethyl-7-[2-methyl-6-(1H-1, 2, 4-triazole-3-yl) pyridine-3-yl]-3, 5-dihydropyrazino [2, 3-b] pyrazine-2 (1H)-ketone, and the structural formula of the CC-115 is shown in the description. An in-vitro Vero cell infection model finds that the CC-115 can inhibit the replication of a new coronavirus (SARS-CoV-2) original strain and an Omicro variant (EG.5.1), and the IC50 (half maximal inhibitory concentration) of the CC-115 aiming at the SARS-CoV-2 original strain is 0.004 [mu] M and 0.005 [mu] M. The CC-115 has the advantages that the CC-115 can inhibit the replication of the SARS-CoV-2 original strain and the Omicro variant (EG.5.1); the CC-115 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 CC-115 in the preparation of anti-new coronavirus drugs. Background Art

[0002] 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 potentially 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 CC-115 has the activity of inhibiting the replication of the new coronavirus by screening the kinase inhibitor library.

[0004] CC-115 is a dual inhibitor of the DNA-dependent protein kinase DNA-PK and the mammalian target of rapamycin mTOR (mTORC1 and mTORC2), with IC50 values ​​of 0.013 μM and 0.021 μM, respectively. DNA-dependent protein kinase (DNA-PK) is a key component of the DNA damage repair machinery and is crucial for maintaining genomic integrity. mTOR is a key mediator of the phosphoinositide 3-kinase / protein kinase B (PI3K / Akt) pathway, playing important roles in cell proliferation, apoptosis, and metabolism. CC-115 has potential anti-tumor activity. In vitro studies have shown that CC-115 inhibits the proliferation of PC-3 human prostate cancer cells with an IC50 value of 138 nM. CC-115 exhibits favorable in vivo pharmacokinetic profiles across multiple species, with oral bioavailability of 53%, 76%, and ~100% in mice, rats, and dogs, respectively. In mouse studies, CC-115 was tested at lower doses of 0.25, 0.5, and 1 mg / kg bid or 1 mg / kg qd, and corresponding tumor volume reductions of 46%, 57%, 66%, and 57%, respectively, were observed (Mortensen DS, et al. Optimization of a Series of Triazole Containing Mammalian Target of Rapamycin (mTOR) Kinase Inhibitors and the Discovery of CC-115. J Med Chem. 2015 Jul 23; 58(14): 5599-5608.). In patients with chronic lymphocytic leukemia (CLL), CC-115 reduced lymphadenopathy (Thijssen R, et al. Dual TORK / DNA-PK inhibition blocks critical signaling pathways in chronic lymphocytic leukemia. Blood. 2016 Jul 28; 128(4): 574-83.). CC-115 has demonstrated safety in both in vitro and in vivo studies and is suitable for clinical drug development. However, the therapeutic effect of CC-115 in SARS-CoV-2 infection has not been studied. The present study found that CC-115 can inhibit the replication of SARS-CoV-2, but the specific mechanism requires further investigation. Summary of the Invention

[0005] The purpose of the present invention is to provide the use of CC-115 in the preparation of anti-new coronavirus drugs. The new coronavirus is SARS-CoV-2, and the CC-115 includes its pharmaceutically acceptable salts or derivatives. The chemical name of CC-115 is: 1-ethyl-7-[2-methyl-6-(1H-1,2,4-triazol-3-yl)pyridin-3-yl]-3,5-dihydropyrazino[2,3-b]pyrazin-2(1H)-one, the molecular formula is: C16H16N8O, and the structural formula is as follows:

[0006] The present invention reveals that CC-115 has the effect of inhibiting the replication of the new coronavirus. Through in vitro Vero cell infection model studies, it was shown that CC-115 can inhibit the replication of the original strain of the new coronavirus (SARS-CoV-2) and the Omicron variant (EG.5.1), and has anti-new coronavirus activity. In the Vero cell infection model, the IC50 against the original strain of SARS-CoV-2 was 0.004μM, and the IC50 against the Omicron variant (EG.5.1) was 0.005μM. CC-115 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, CC-115 can significantly inhibit the replication of the new coronavirus, with an IC50 of 0.004μM against the original strain of SARS-CoV-2 and an IC50 of 0.005μM against the Omicron variant (EG.5.1). 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] Vero cells were cultured in MEM medium at 37°C in a 5% CO2 incubator. The day before drug screening, an appropriate number of Vero cells were seeded into 48-well plates, allowing the cells to reach a density of over 90% the next day. A library of small molecule kinase inhibitors was purchased from a commercial company at an initial concentration of 10 mM. The cells were diluted to a 100 μM initial concentration in MEM medium. The cells were then diluted to 10 concentrations using a 3.33- and 3-fold cross-over series, resulting in drug concentrations of 30, 10, 3, 1, 0.3, 0.1, 0.03, 0.01, 0.003, and 0.001 μM, respectively. For each concentration, 500 μL of drug-containing medium was added to the Vero cells. Next, SARS-CoV-2 infection was performed in a biosafety level 3 laboratory. The original virus solution was diluted in MEM medium at appropriate ratios, and 10 μL of the diluted solution was added to the culture wells to be infected, achieving a final infection titer of approximately 100 times the TCID50. A negative control with no virus infection and a positive control with virus but no drug were also performed, each in triplicate. 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 In this case, 100 μM and 30 μM CC-115 caused cytopathic effects with obvious morphological changes, and although they inhibited viral replication, they were not counted as effective concentrations.

[0013] In summary, the present invention provides a new use of CC-115 against COVID-19. CC-115 is a potential drug for treating COVID-19 infection.

Claims

1. The use of CC-115 in the preparation of anti-new coronavirus drugs, characterized in that: The CC-115 includes pharmaceutically acceptable salts thereof. The chemical name of CC-115 is: 1-ethyl-7-[2-methyl-6-(1H-1,2,4-triazol-3-yl)pyridin-3-yl]-3,5-dihydropyrazino[2,3-b]pyrazin-2(1H)-one, the molecular formula is: C16H16N8O, 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 CC-115 exhibits anti-COVID-19 activity by inhibiting the replication of the original SARS-CoV-2 strain and the Omicron variant EG.5.1.

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