Proteolytic cleavage of influenza A virus (IAV) hemagglutinin by host proteases is crucial for virusinfectivity and spread. The transmembrane serine protease TMPRSS2 was previously identified as the essential protease that can cleave hemagglutinin of many subtypes of influenza virus and spike protein of coronavirus. Herein, we found that a guanine rich tract, capable of forming intramolecular G-quadruplex in the presence of potassium ions, in the promoter region of human TMPRSS2 gene was quite important for gene transcriptional activity, hence affecting its function. Furthermore, 7 new synthesized benzoselenoxanthene analogues were found to enable stabilizing such G-quadruplex.
More importantly, compounds can down-regulate TMPRSS2 gene expression, especially endogenous TMPRSS2 protein levels, and consequently suppress influenza A virus propagation in vitro. Our results provide a new strategy for anti-influenza A virus infection by small molecules targeting the TMPRSS2 gene G-quadruplex and thus inhibiting TMPRSS2 expression, which is valuable for developing small molecule drugs against influenza A virus and also may be a potential candidate as anti-SARS-CoV-2 (Severe Acute Respiratory Syndrome CoV 2) lead molecules.Figure 5. Benzoselenoxanthene analogues inhibit influenza virus propagation. (A) Compounds inhibit IAV (A/ PR8/34) in vitro.Firstly, Calu-3 cells were treated with compounds or Oseltamivir and Camostat as references for 24 h, then, infected with IAV and followed by culture in compounds contained medium, finally, supernatants were collected at 48 h after infection and viral titers were determined by plaque assay. (B) Compounds suppressed cytopathic effect. After Calu-3 cells were treated with compounds (8 μM) and infected with IAV (A/ PR8/34, herein, abbr.: PR8) as described above, photos were collected at 48 h after infection. (C) The inhibitory effects of Se1, Se3 and Se5 on IAV spread can be rescued by trypsin. Firstly, Calu-3 cells were treated with compounds or Oseltamivir as references for 24 h, then, infected with IAV and followed by culture in compounds and trypsin (4 μg/mL) contained medium, finally, supernatants were collected at 48 h after infection and viral titers were determined by plaque assay. All of experiments were repeated three times. Statistical significance was determined using Student's two-tailed t-test (A,C). *P < 0.05, **P < 0.01, ***P < 0.001. Error bars represent the SEM of three independent experiments. Scientific RepoRtS | (2020) 10:7635 | https://doi.PCR stop assay. PCR reaction system contained 1 μM of TMPRSS2-L and TMPRSS2-Rev, 0.2 mM dNTP, 0.1 U/μL taq polymerase and different concentrations of compounds. PCR setting was as follow: 94 °C for 5 min, followed by 10 cycles of 94 °C for 30 s, 60 °C for 30 s, and 72 °C for 30 s. Amplified products were run on 12% native PAGE and stained by SYBR Gold.