Human alveolar adenocarcinoma (A549) cells and Vero E6 cells were purchased from the American Type Culture Collection (United States) and cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS) at 37 °C in a 5% CO₂ humidified incubator. HTNV 76-118 was donated by researcher Changshou Hang, Chinese Center for disease Control and Prevention (CDC), and was preserved and expanded by our laboratory. HTNV strain 76–118 was propagated in Vero E6 cells. The titers of HTNV were measured by immunofluorescence assay (IFA). Median tissue culture infective dose (TCID50) of the virus was determined by IFA through the Reed-Muench formula. TCID50 and Plaque Forming Units (PFUs) are converted by the following formula, 1 PFUs≈0.7 TCID50. For infection, A549 cells were rinsed with DMEM, and cells were infected at a multiplicity of infection (MOI) of 1. All experiments with the virus were performed in a biosafety level 2 facilities, in accordance with the institutional biosafety operating procedures.

The compounds were synthesized and characterized according to the method reported in our previous studies (Li et al., 2015; Qu et al., 2020), in briefly, a mixture of aromatic aldehyde (10 mmol) and 4-hydroxycoumarin (20 mmol), or A mixture of 3,5-cyclohexanedione (10 mmol), 3-cyanobenzaldehyde (10 mmol), malononitrile (10 mmol) and 4-(dimethylamino) pyridine (DMAP) (1 mmol) was dissolved in 100 ml of ethanol. A few drops of piperidine were added, and the mixture was stirred for 3 h at room temperature. After the completion of reaction as determined by TLC, water was added until precipitation occurred. After filtering the precipitates, all the compounds were sequentially washed with ice-cooled water and ethanol and then dried under a vacuum. Infrared spectra were measured using a Brucker Equinox-55 spectrophotometer (Bruker Optics, Ettlingen, German). ¹H NMR spectra, ¹³C NMR spectra, and mass spectra were tested using the Varian Inova-400 spectrometer (Varian Inc, CA, United States), Bruker Avance III spectrometer (Bruker Optics), and micrOTOF-Q II mass spectrometer (Bruker Optics), respectively (Schemes 1, 2).

A549 cells were seeded in 96-well plates at a density of 1×10⁴ cells in a total volume of 100 μL per well and incubated overnight at 37°C and 5% CO₂. Cells with a confluence of 70–80% were treated with the coumarin derivatives at a final concentration of 10 μM or DMSO (0.1%) with infected HTNV at an MOI of 1. After incubating for 4 h at 37°C, the supernatant was discarded, and the cells were cultured in DMEM supplemented with 2% (v/v) FBS at 37 °C in a 5% CO₂.

HTNV-infected cells were rinsed with PBS after 96 h post infection (hpi) and fixed with ice-cold 4% paraformaldehyde in PBS for 30 min at RT, followed by the treatment of 0.1% Triton X-100 for 20 min. Then, cells were blocked with 3% bull serum albumin (BSA) in PBS at 37°C for 1 h and stained with mouse anti-HTNV NP monoclonal antibodies 1A8 (1:1,000, prepared by our lab) at 37°C for 1 h. The pre-stained cells were incubated with Alexa Fluor Cy3-conjugated anti-mouse IgG (1:400) at 37°C for 1 h and stained with DAPI (1:5,000, Sigma). Samples were observed using an inverted fluorescence microscope (Olympus, Japan). The intensity was measured by an Infinite 200 PRO microplate reader (TECAN, Switzerland).

The signals of DAPI and NP corresponded to the number of live cells and extensiveness of HTNV infection in each well, respectively. The percentage of infection was determined by the following formula: NP signal count/DAPI signal count×100%. Subsequently, the percentage inhibition of viral replication was determined as Inhibition rate (%) = [(Ac−As)/(Ac−Ab)]×100, in which Ac referred to the infection rate of vehicle controls, whereas referred to HTNV-infected cells treated with the coumarin derivatives, and Ab referred to the blank control value of uninfected and IFA-treated cells. The inhibition rate of coumarin derivatives more than 80% was selected for further research. The Z′ factor was calculated using the following equation and determined to be 0.54, indicating that the primary screening was robust (Zhang et al., 1999). Z ' = 1 − ( 3 σ c + + 3 σ c − ) | μ c + − μ c − |

Cell viability was examined using the cell counting kit-8 (CCK-8) assay (YEASEN, China). In brief, A549 cells (1×10⁴ cells/well) in confluent 96-well cell culture plates were treated with different concentrations of coumarin derivatives (1, 2.5, 5, 10, 25, 50, and 100 μM) for 48 h. Then, 10 μL of CCK-8 reagents was added into each well for 2 h of incubation. Optical density was detected using the microplate reader (BioTek, Germany). The values were then fit into a non-linear regression curve, and CC₅₀, which is the concentration that results in 50% cell viability, was calculated using GraphPad Prism by interpolation.

Cells were harvested, and the total cellular mRNA was extracted by the RNA extraction kit (Axygen, United States) according to the manufacturer’s procedures. Reverse transcription (RT) was performed using Prime Script RT Master Mix (YEASEN, China). The quantity of RNA was determined by the QuantiTect SYBR Green RT-PCR kit (YEASEN, China), and the sequence of primer was as follows: HTNV-S F-GAGCCTGGAGACCATCTG, R-CGGGACGACAAAGGATGT; β-actin F-5′-TGACGGGGTCACCCACACTG-3′, R-5′-AAGCTGTAGCCGCGCTCGGT-3′). cDNA was denatured at 95°C for 30 s and amplified for 45 cycles of 10 s at 95°C, 31 s at 60°C in LightCycler 96 (Roche, Switzerland). The mRNA expression level of the target gene was normalized to the β-actin. Relative values were calculated using the ^∆∆Ct method.

A549 cells infected with HTNV at an MOI of 1.0 were treated by coumarin derivative and washed with Tris-buffered saline (TBS). At 96 hpi, cells were lysed by adding 100 μL of RIPA Lysis Buffer System containing protease and phosphatase inhibitors. Then, cell extraction was subjected to gel electrophoresis and transferred onto a polyvinylidene difluoride membrane (Millipore, Germany). After blocking with 3% BSA in TBS for 2 h, the membrane was probed with primary antibodies overnight at 4°C; the antibody against HTNV nucleus protein was diluted at 1:1,000, and the antibody for GAPDH (Abcam, UK) was diluted at 1:1,000. After secondary antibodies labeled with infrared dyes were added, the signals were visualized using the Odyssey Infrared Imaging System (Biosciences, United States).

Three-day old neonatal mice were provided by the Experimental Animal Center of Fourth Military Medical University and randomly divided into three groups, namely, the vehicle (corn oil) treatment group, HTNV infection group, and the HTNV infection with coumarin treatment group. The body weight of the mice was measured from 0 days post infection (dpi) to 15 dpi. At 13 dpi, the tissues of mice, including the lungs, livers, kidneys, and brains, were obtained for hematoxylin and eosin (H&E) staining and qRT-PCR assay. All animal experiments were authorized by the ethical committee of the Fourth Military Medical University.

Search for the corresponding protein model in the PDB database (Protein Date Bank, https://www.rcsb.org/, PDB) according to the predicted target, and set the docking domain based on the active protein site described in the literature. Then use AutoDock Tools (1.5.6) to perform semi-flexible molecular docking. Finally, used Discovery studio 2019 to visualize the results. The structure of Akt1 protein was modeled with reference to PDB database (PDB ID: 4EKL). Based on the published literature (Lin et al., 2012), the active center is x = 28.03, y = 5.22, z = 10.89, and the semi-flexible docking result shows that the minimum active free energy is −9.35 kcal/mol, exhibiting a good affinity.

All data were expressed as the mean ± standard errors. One-way analysis of variance (ANOVA), two-way ANOVA, and Kaplan–Meier survival analysis were used for statistical evaluations. Statistical analysis was performed using Student’s t-test. A p value of <0.05 was considered statistically significant. The dose–response curve was created by the nonlinear regression model, and EC₅₀ and CC₅₀ were calculated using GraphPad Prism 5.0 software.

We synthesized and observed the bioactivity of six series of coumarin derivatives to identify the possible coumarin-based small-molecule chemical compounds with anti-HTNV activity. Among the 126 compounds, two biscoumarin derivatives (N6 and N7) showed a potent inhibitory activity against HTNV by a high-throughput screen (HTS) assay, and the percentage of inhibition of N6 and N7 was 85.4 and 83.1%, respectively ( Figure 1A, B ). Compared with the non-infected A549 cells and vehicle-treated HTNV-infected A549 cells, the inhibitory activity of N6 and N7 were validated by IFA ( Figure 1C ). N6 and N7 belonged to the biscoumarin chemical structure ( Figure 1D ), which was characterized by ¹H nuclear magnetic resonance (NMR), ¹³C NMR, DEPT135, ¹H–¹H COSY, HSQC, HMBC, NOESY, and high-resolution mass spectrometry ( Supplementary Figure S1 ). Furthermore, the qRT-PCR results indicated that N6 and N7 could significantly inhibit the expression of HTNV-S genes ( Figure 1E ).

The selectivity index (SI) is a ratio that compares drug’s cytotoxicity and antiviral activity, and a high SI indicates that a drug is safe and effective in the clinic. Thus, we observed the half cytotoxicity concentration (CC₅₀) of N6 and N7 to the A549 cells determined by CCK-8 staining. The results showed that CC₅₀ of N6 and N7 was 77.08 ± 0.56 μM and 64.78 ± 0.53 μM, respectively ( Figure 2A, B ). Then, we measured the inhibitory effects of N6 and N7 at the concentration ranging from 1 to 50 μM on HTNV (MOI = 1). Both coumarin derivatives displayed an anti-HTNV activity in a dose-dependent manner, and EC₅₀ of N6 and N7 was 7.06 ± 1.05 μM and 11 ± 0.76 μM, respectively ( Figure 2C ). Based on the value of CC₅₀ and EC₅₀, the SI of N6 and N7 was 10.9 and 5.9, respectively. N6 had a potent inhibitory activity against HTNV and less toxicity to A549 cells in vitro. Thus, we treated the HTNV with 10 μM N6 for 2 h at RT or 37°C to confirm whether N6 had a direct virucidal effect on the HTNV and then infected the A549 cells. Contrary to the negative control (NC), the Western blot assay showed that N6-treated HTNV could not affect the expression of HTNV NP at 96 hpi ( Figure 2D ). This result indicated that N6 inhibited the HTNV through the indirect virucidal activity.

Considering that N6 presented an inhibitory ability against HTNV without the direct virucidal activity, we hypothesized that this compound might inhibit the replication of HTNV in infected A549 cells. Then, the viral titer and expression of HTNV protein in HTNV-infected A549 cells were measured after N6 treatment at 96 hpi. Compared with the untreated control cells, the viral titer of HTNV was reduced significantly after 5, 10, and 20 μM N6 treatments ( Figures 3A,B ). Meanwhile, qRT-PCR assay showed that 5, 10, and 20 μM N6 treatments significantly inhibited the expression of HTNV-S RNA in a dose-dependent model ( Figure 3C ), whereas only 10 and 20 μM N6 treatments could reduce the expression of HTNV-NP ( Figure 3D, E ).

We designed seven different administration regimens of N6 in HTNV-infected A549 cells to explore the possible administration regimen of N6 against HTNV infection ( Figure 4A ). Compared with the uninfected cells (NC), the expression of HTNV-NP was only reduced efficiently in three administration regimens of N6 in HTNV-infected A549 cells, namely, the regimen of 2 h pre-infection to 96 hpi (lane d), from 2 hpi to 96 hpi (lane e), and from 12 hpi to 96 hpi (lane f). These results suggested that the administration of N6 couldn’t influence HTNV binding and entry (lane b and c). According to the results of lane e and f, N6 mainly exerts antiviral effect at the early stage of HTNV replication. Meanwhile, lane g and h have less anti-HTNV effect, which indicates that N6 has less effect on the middle and late stages of HTNV replication ( Figures 4A–C ). In summary, N6 at the early stage of HTNV replication could inhibit the replication and production of infectious HTNV in host cell.

Based on the effective administration of N6 regimens against HTNV infection in vitro, we evaluated the therapeutic efficacy of N6 in HTNV-infected newborn mice at the early stage of infection. In contrast to the vehicle-treated HTNV-infected mice group (HTNV), N6 treatment (N6-HTNV) could prolong the survival rate of HTNV-infected mice from 60 to 80% ( Figure 5A ) and improve the weight loss of HTNV-infected mice during the 15-days therapy ( Figure 5B ). Furthermore, compared with the infected group (HTNV), the N6 treatment group could reduce the expression of HTNV-S RNA in the brain, liver, lung, and kidney ( Figure 5C ).

Pathological change was also observed in HTNV-infected mice after N6 treatment. At 14 dpi, the tissues of mice from the control and N6 treatment groups were collected, and the H&E staining showed that the swelling of liver cells and hepatic sinusoidal dilation and congestion were observed in the HTNV infection model group, whereas these pathological changes were less pronounced in the N6-treated group. The normal alveolar structure disappeared in HTNV-infected mice in the model group, and a large number of neutrophils were accumulated in the lung tissue, whereas the alveolar fusion was alleviated in N6-treated mice ( Figure 5D ). However, no evident pathological change was observed in the brain and kidney in HTNV-infected mice after N6 treatment ( Supplementary Figure S2 ).

Increasing evidence showed that phosphoinositide kinases played a momentous role in mediating viral infections (Burke, 2018), and the coumarin derivatives exhibited a tremendous inhibitory activity toward the phosphoinositide 3-kinase (PI3K) and protein kinase B (Akt) pathways (Ma and Liu, 2017; Umar et al., 2020). We also explored the possible role of N6 targeting the PI3K/Akt signaling pathway during HTNV infection. The molecular docking results showed that N6 formed interactions with the key amino acid residues at its active site and revealed several molecular interactions responsible for the observed affinity: 1) two conventional hydrogen bond interactions between the hydroxyl group of the sulfonyl group and fluorine atom with GLU234 and ASP439; 2) pi-alkyl interactions between the benzopyrone ring and LEU156, ALA177, ALA230, and PHE442; 3) pi-sigma interactions between the benzopyrone ring and VAL164 and MET 281; and 4) pi-sulfur interaction between the benzopyrone ring and GLU278 ( Figure 6A, B ). In addition, the binding surface model of N6 and Akt1 was analyzed, including the aromatic ring edges or faces, hydrophobicity, hydrogen bond, ionizability, atomic charge, and solvent accessibility surface ( Figure 6C, H ). Considering that the allosteric inhibitors could inactivate the phosphorylated conformation of Akt1, we performed Western blot to observe the effect of N6 on the expression of phosphorylated Akt1. Compared with the vehicle (DMSO) or N6 treatment in control A549 cells, the expression of p (Ser473)Akt and HTNV-NP was augmented in HTNV-infected cells, and the treatment of N6 could inhibit the expression of p (Ser473)Akt and HTNV-NP significantly ( Figure 7).