Application of micromolecular drug TUG-1375 targeting host factor FFAR2 in resisting influenza virus infection
The small molecule drug TUG-1375, which targets the host factor FFAR2, solves the problem of the lack of effective inhibitory drugs for different subtypes of influenza viruses in the existing technology, and achieves effective inhibition of SH13 (H9N2) and FZ09 (H1N1) influenza viruses, especially by inhibiting the expression of M1 protein, demonstrating its potential for application in anti-influenza virus.
Patent Information
- Application Number
- CN202511114167.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-08-11
- Publication Date
- 2025-10-17
AI Technical Summary
The existing technology lacks effective small molecule drugs against different subtypes of influenza viruses, especially the inhibition of SH13 (H9N2) and FZ09 (H1N1) influenza viruses, and commonly used antiviral drugs face the problem of strain resistance.
Develop a small molecule drug TUG-1375 targeting the host factor FFAR2 for the preparation of anti-influenza virus drugs, which blocks influenza virus replication by inhibiting the function of the host factor FFAR2.
TUG-1375 can significantly inhibit the replication of SH13 (H9N2) and FZ09 (H1N1) influenza viruses, especially by inhibiting the expression of the late protein M1, showing potential application value against different subtypes of influenza viruses.
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Figure CN120789059A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The application relates to application of a small-molecule drug TUG-1375 targeting a host factor FFAR2 as an antiviral drug in anti-influenza virus infection, and belongs to the field of biological medicine. BACKGROUND
[0002] Influenza virus belongs to Orthomyxoviridae, and is an influenza virus with a diameter of 80-120 nm and composed of 8 single-strand negative-strand RNAs. After influenza virus infection, the epithelial cells of the respiratory mucosa are mainly invaded, causing epithelial cell necrosis and mucosal hyperemia and edema. The infection of the host is extensive, including poultry, birds, pigs, and can also cross the interspecies barrier to infect humans, causing great threat to poultry industry and human health, such as the 1918 Spanish H1N1 influenza, the 2009 H1N1 influenza, H3N8, H5N6, H5N8, H7N9 and H10N3 avian influenza virus subtypes. At present, the prevention and control of influenza virus mainly depends on vaccine immunization and drug treatment. However, the continuous emergence of new subtypes of strains makes us have to update the vaccine regularly; at the same time, the long-term application of commonly used antiviral drugs urgently needs to solve the problem of drug resistance of strains. In the process of infecting host cells, influenza virus needs a series of host factors to assist in completing the process. Therefore, the development of small-molecule drugs targeting host factors is a good alternative strategy.
[0003] G protein-coupled receptor (GPCR) is the largest receptor family on the cell membrane surface, and there are about 800-1000 in the human genome, accounting for about 50% of all cell membrane receptors. GPCR family proteins are key target molecules for cells to perceive extracellular stimuli and transmit signals inward, and they mainly regulate intracellular physiological functions through downstream signal molecules such as G proteins. So far, about 30% of GPCR family proteins are known as drug development targets; in virology research, GPCR family proteins (FFAR2, mGluR2, 5-HT, mAChR, CXCR4) are involved in regulating the invasion process of influenza, rabies, Ebola, Marburg, AIDS and other viruses. Our previous research found that FFAR2, a low-chain fatty acid receptor subfamily in the GPCR family, can promote the endocytosis of influenza virus. Small-molecule drug TUG-1375 is a targeting FFAR2 agonist, and its structure is as follows:
[0004]
[0005] Small-molecule drug TUG-1375 has no effect on FFAR3, FFAR4, PPARa, PPARy, PPARd, LXRa and LXRb. There is no research report on the effect of small-molecule drug TUG-1375 on influenza virus. Therefore, we explore the effect of TUG-1375 on different subtypes of influenza virus after treating A549 cells. SUMMARY
[0006] The technical problem to be solved by the present application is to provide a new use of a small molecule drug TUG-1375 in resisting influenza virus.
[0007] The technical solution of the present application is the use of a small molecule drug TUG-1375 in preparing an anti-influenza virus drug, wherein the influenza virus is A / chicken / Shanghai / SC197 / 2013(H9N2) avian influenza virus or A / Fuzhou / 1 / 2009(H1N1) influenza A virus.
[0008] Compared with the prior art, the present application has the following beneficial effects:
[0009] The present application first detects the influence of the small molecule drug TUG-1375 on the replication of different subtypes of influenza virus after treating A549 cells, and then explores the influence of the small molecule drug TUG-1375 on the expression of FZ09(H1N1) influenza virus protein. The plaque titration result shows that the small molecule drug TUG-1375 has no influence on the replication of WSN(H1N1) and AH05(H5N1) viruses, but can inhibit the replication of SH13(H9N2) and FZ09(H1N1) influenza viruses. The Western blotting result shows that the small molecule drug TUG-1375 can inhibit the expression of FZ09(H1N1) influenza virus late protein M1 and further inhibit the replication of influenza virus, further consolidating the important regulatory function of host factor FFAR2 in regulating influenza virus replication, which can become an ideal drug target for resisting influenza virus replication and has potential application value. BRIEF DESCRIPTION OF DRAWINGS
[0010] Figure 1 A549 cell toxicity detection of different concentrations of TUG-1375.
[0011] Figure 2 TUG-1375 has no influence on the replication of WSN(H1N1) influenza virus.
[0012] Figure 3 TUG-1375 has no influence on the replication of AH05(H5N1) highly pathogenic influenza virus.
[0013] Figure 4 TUG-1375 inhibits the replication of SH13(H9N2) influenza virus.
[0014] Figure 5 TUG-1375 inhibits the replication of FZ09(H1N1) influenza A virus.
[0015] Figure 6TUG-1375 inhibits the expression of M1 protein of FZ09 (H1N1) influenza A virus. DETAILED DESCRIPTION
[0016] The experimental methods in the following examples are all conventional methods unless otherwise specified. The experimental materials used in the following examples are all purchased from commercial channels unless otherwise specified.
[0017] Experimental materials: A549 cells (human lung cancer epithelial cells) were purchased from ATCC; small molecule compound TUG-1375 (MCE, HY-112813) was purchased from MCE company and dissolved in DMSO, prepared into 100 mM stock solution, and stored at -80°C. Figure 1
[0018] Influenza viruses: A / WSN / 33 (H1N1), abbreviated as WSN (H1N1); A / Fuzhou / 1 / 2009 (H1N1), abbreviated as FZ09 (H1N1); A / Anhui / 2 / 2005 (H5N1), abbreviated as AH05 (H5N1); A / chicken / Shanghai / SC197 / 2013 (H9N2), abbreviated as SH13 (H9N2); these strains are strains published before the application date and propagated and preserved by the laboratory. The experiments related to highly pathogenic H5N1 subtype influenza viruses in this experiment were carried out in the P3 laboratory of Harbin Veterinary Research Institute of Chinese Academy of Agricultural Sciences, which was approved by the Ministry of Agriculture and China National Accreditation Service (CNAS).
[0019] Example 1: Determination of the effect of different concentrations of TUG-1375 on the activity of A549 cells by CellTiter Glo cell viability kit
[0020] A549 cell suspension was plated in a 96-well plate and placed in a 37°C cell incubator (5% CO2) for culture; 100 mM TUG-1375 stock solution was diluted with F-12K medium (0.3% BSA) to 200, 150, 100 mM working solution, respectively, and then 150 μL of TUG-1375 working solution and DMSO diluent of different concentrations (three wells each) were added to the 96-well plate with A549 cell density of 95%. After incubation in a 37°C cell incubator for 24 h, 100 μL of CellTiter-Glo solution (G7572, Promega) was added to each well and incubated at 37°C for 10 min. The fluorescence value was measured by GloMax 96 microplate luminometer (Promega). The experimental data was plotted by Graphpad Prism 8 software and statistically analyzed.
[0021] Results as shown in Figure 1 As shown in Table 1, 200 μΜ TUG-1375 produced nearly 15.52% inhibition of A549 cell viability compared to the negative control group, and 150 and 100 μΜ TUG-1375 had no toxic effect on A549 cell viability.
[0022] Example Two, TUG-1375 has no effect on WSN (H1N1) influenza virus replication
[0023] A549 cell suspension was plated in 12-well plates and incubated in a 37°C cell incubator (5% CO2). 100 mM TUG-1375 stock solution was diluted with F-12K medium (0.3% BSA) to 150 and 100 μΜ working solutions, respectively, and then 1 mL of 150 and 100 μΜ TUG-1375 working solution and DMSO diluent (three wells each) were added to the 12-well plates with A549 cell density of 95%. After 3 h of drug pretreatment, A549 cells were infected with WSN (H1N1) virus (MOI = 0.01), and after 1 h of infection, the cells were washed twice with sterile PBS, 1 mL of 150 and 100 μΜ TUG-1375 working solution was added to the corresponding wells, respectively, and incubated in a 37°C cell incubator with 5% CO2. Supernatant was collected at 12 and 24 h after WSN (H1N1) virus infection, respectively, and then plaque titration was performed.
[0024] The results of plaque titration are shown in Table 2. 150 and 100 μΜ TUG-1375 had no effect on WSN (H1N1) virus replication at 12 and 24 h after virus infection. Figure 2
[0025] Example Three, TUG-1375 has no effect on highly pathogenic AH05 (H5N1) influenza virus replication
[0026] A549 cell suspension was plated in 12-well plates and incubated in a 37°C cell incubator (5% CO2). 100 mM TUG-1375 stock solution was diluted with F-12K medium (0.3% BSA) to 150 and 100 μΜ working solutions, respectively, and then 1 mL of 150 and 100 μΜ TUG-1375 working solution and DMSO diluent (three wells each) were added to the 12-well plates with A549 cell density of 95%. After 3 h of drug pretreatment, A549 cells were infected with AH05 (H5N1) virus (MOI = 0.1), and after 1 h of infection, the cells were washed twice with sterile PBS, 1 mL of 150 and 100 μΜ TUG-1375 working solution was added to the corresponding wells, respectively, and incubated in a 37°C cell incubator with 5% CO2. Supernatant was collected at 12 and 24 h after AH05 (H5N1) virus infection, respectively, and then plaque titration was performed.
[0027] The results of plaque titration are shown in Table 2. Figure 3 As shown in Table 2, 150 and 100 μΜ TUG-1375 had no effect on the replication of AH05 (H5N1) virus at 12 and 24 h post-virus infection.
[0028] Example Four, TUG-1375 Inhibits SH13 (H9N2) Influenza Virus Replication
[0029] A549 cell suspension was plated in 12-well plates and incubated in a 37 °C cell incubator (5% CO2). 100 mM TUG-1375 stock solution was diluted with F-12K medium (0.3% BSA) to 150 and 100 μΜ working solutions, respectively, and then 1 mL of 150 and 100 μΜ TUG-1375 working solution and DMSO diluent (three wells for each) were added to the 12-well plates with A549 cell density of 95%. After 3 h of drug pretreatment, A549 cells were infected with SH13 (H9N2) virus (MOI = 0.1), and 1 h after infection, the cells were washed twice with sterile PBS, and 1 mL of 150 and 100 μΜ TUG-1375 working solution was added to the corresponding wells, respectively, and incubated in a 37 °C cell incubator with 5% CO2. The supernatant was collected at 12 and 24 h post-SH13 (H9N2) virus infection, respectively, and then plaque titration was performed.
[0030] The results of plaque titration are shown in Table 2. Figure 4 As shown in Table 2, 150 and 100 μΜ TUG-1375 had no effect on the replication of AH05 (H5N1) virus at 12 and 24 h post-virus infection.
[0031] Based on the above results, it can be concluded that TUG-1375 can dose-dependently inhibit the replication of SH13 (H9N2) avian influenza virus.
[0032] Example Five, TUG-1375 Inhibits FZ09 (H1N1) Influenza A Virus Replication
[0033] A549 cell suspension was plated in 12-well plates and incubated in a 37°C cell incubator (5% CO2). 100 mM TUG-1375 stock solution was diluted with F-12K medium (0.3% BSA) to 150 and 100 μM working solutions, respectively. Then 1 mL of 150 and 100 μM TUG-1375 working solution and DMSO diluent (three wells for each) were added to the 12-well plates with 95% cell density. After 3 h of drug pre-treatment, A549 cells were infected with FZ09 (H1N1) virus (MOI = 0.1). After 1 h of infection, the cells were washed twice with sterile PBS, and 1 mL of 150 and 100 μM TUG-1375 working solution was added to the corresponding wells, respectively. The plates were incubated in a 37°C cell incubator with 5% CO2. The supernatant was collected at 12 and 24 h after FZ09 (H1N1) virus infection, respectively, and then subjected to plaque titration.
[0034] The results of plaque titration are shown in Figure 5 150 μM TUG-1375 inhibited FZ09 (H1N1) virus replication by 4.73-fold and 3.61-fold at 12 and 24 h, respectively; 100 μM TUG-1375 inhibited FZ09 (H1N1) virus replication by 2.57-fold and 3.07-fold at 12 and 24 h, respectively.
[0035] Example Six, TUG-1375 Inhibits the Expression of M1 Protein of FZ09 (H1N1) Influenza A Virus
[0036] A549 cell suspension was plated in 12-well plates and incubated in a 37°C cell incubator (5% CO2). 100 mM TUG-1375 stock solution was diluted with F-12K medium (0.3% BSA) to 150 and 100 μM working solutions, respectively. Then 1 mL of 150 and 100 μM TUG-1375 working solution and DMSO diluent (three wells for each) were added to the 12-well plates with 95% cell density. After 3 h of drug pre-treatment, A549 cells were infected with FZ09 (H1N1) virus (MOI = 0.1). After 1 h of infection, the cells were washed twice with sterile PBS, and 1 mL of 150 and 100 μM TUG-1375 working solution was added to the corresponding wells, respectively. The plates were incubated in a 37°C cell incubator with 5% CO2. The supernatant was collected at 12 and 24 h after FZ09 (H1N1) virus infection, respectively, and then subjected to plaque titration.
[0037] The results of western blotting are shown in Figure 6As shown, when the virus-infected A549 cells were treated with 150 μM TUG-1375, no effect on the early expressed proteins PB2, PB1 and NP and the late expressed protein M2 of the influenza virus was observed, but the expression of the late protein M1 was inhibited.
[0038] The primary antibodies used in the Western blotting experiment: PB2, PB1 and NP antibodies (monoclonal antibodies prepared in the laboratory), M1 antibody (GTX125928, GeneTex), M2 antibody (GTX125951, GeneTex), GAPDH (60004-1-Ig, Proteintech).
[0039] In summary, the results show that TUG-1375 can inhibit the replication of FZ09 (H1N1) influenza A virus by inhibiting the expression of M1 protein.
Claims
1. Use of the small molecule drug TUG-1375 in the preparation of an anti-influenza virus drug, wherein the influenza virus is A / chicken / Shanghai / SC197 / 2013 (H9N2) avian influenza virus or A / Fuzhou / 1 / 2009 (H1N1) influenza A virus.