Broad-spectrum antiviral activity of Spatholobus suberectus Dunn against SARS-CoV-2, SARS-CoV-1, H5N1, and other enveloped viruses

Abstract

The current COVID-19 pandemic caused by SARS-Cov-2 is responsible for more than 6 million deaths globally. The development of broad-spectrum and cost-effective antivirals is urgently needed. Medicinal plants are renowned as a complementary approach in which antiviral natural products have been established as safe and effective drugs. Here, we report that the percolation extract of Spatholobus suberectus Dunn (SSP) is a broad-spectrum viral entry inhibitor against SARS-CoV-1/2 and other enveloped viruses. The viral inhibitory activities of the SSP were evaluated by using pseudotyped SARS-CoV-1 and 2, HIV-1_{ADA and HXB2} , and H5N1. SSP effectively inhibited viral entry and with EC₅₀ values ranging from 3.6 to 5.1 μg/ml. Pre-treatment of pseudovirus or target cells with SSP showed consistent inhibitory activities with the respective EC₅₀ value of 2.3 or 2.1 μg/ml. SSP blocked both SARS-CoV-2 spike glycoprotein and the host ACE2 receptor. In vivo studies indicated that there was no abnormal toxicity and behavior in long-term SSP treatment. Based on these findings, we concluded that SSP has the potential to be developed as a drug candidate for preventing and treating COVID-19 and other emerging enveloped viruses.

Keywords: H5N1; HIV-1; SARS-CoVs; Spatholobus suberectus Dunn; antivirals.

FIGURE 1

Flow chart of the study. Preparation and quality‐controlled of SSP which is used to determine the broad‐spectrum antiviral activity and its underlying mechanism. The powder SSP was further extracted to determine the effective active parts

FIGURE 2

Representative pictures of SSP's quality control. (a) HPLC chromatogram of different batches of SSP after ethyl acetate processing. (b) TLC chromatogram of different batches of SSP and positive control of SS

FIGURE 3

Antiviral activity of SSP against SARS‐CoV‐2. Serially diluted SSP was added to HEK293T‐ACE2 cells infected with SARS‐CoV‐2 (a) and VSV (b) respectively. The luciferase level was measured 2 or 3 days post‐infection. To test SSP cytotoxicity, cells viability (c) was measured using the Promega CellTiter‐Glo Luminescent Cell Viability Assay kit. (d) Pre‐treatment of SARS‐CoV‐2 and target cells inhibited viral infection. (e) Binding of RBD to ACE2 expressing 293T cells, but not 293T control cells. (f) SSP inhibited RBD binding to target cells. SSP pre‐treated HEK293T‐ACE2 cells were incubated with RBD‐PD1 for 30 mins on ice, followed by antibody staining of ACE2 (upper panel) and RBD (lower panel) and flow cytometry analysis. The data represent the mean ± SEM of triplicate experiments

FIGURE 4

Antiviral activity of SSP against SARS‐CoV‐1, H5N1, and HIV viruses. Serially diluted SSP was added to HEK293T‐ACE2, MDCK, GHOST‐CCR5, and GHOST‐CXCR4 infected with SARS‐CoV‐1 (a) H5N1 (b) HIV_ADA (c), and HIV_HXB2 (d) respectively. The luciferase level was measured 2 or 3 days post‐infection. To test SSP cytotoxicity, cells viability (e) was measured using the Promega CellTiter‐Glo Luminescent Cell Viability Assay kit. The data represent the mean ± SEM of triplicate experiments

FIGURE 5

Mechanisms of SSP mediated virus entry inhibition. (a ~ b). Pre‐treatment of SARS‐CoV‐1 (a), H5N1 (b) and target cells inhibited viral infection. SARS‐CoV‐1 or H5N1 pseudovirus and HEK293 T‐ACE2 (a) or MDCK (b) cells pre‐treated with serial diluted SSP were recovered and subsequently subjected to infect target cells, or incubated with untreated SARS‐CoV‐1 pseudovirus for 48 hours, respectively. The luciferase level was measured 2 or 3 days post‐infection. (c ~ d). Post‐entry assay. GHOST‐CD4‐CCR5 or CXCR4 cells were coincubated with pseudovirus for 2 hr, washed, and then treated with the presence of 50 mg/ml SSP, dimethyl sulfoxide (DMSO) as the negative control, and 1 mM AZT as a positive control for 48 hr. SSP does not inhibit either HIV‐1_ADA or HIV_HxB2 virus gene replication after the viral entry is achieved. (e ~ f). SSP‐virus interaction assay. HIV‐1_ADA and HIV_HxB2 pseudovirus pre‐treated with 50 mg/ml SSP or DMSO as a negative control and entry inhibitor enfuvirtide (T‐20) as a positive control. SSP pre‐treatment inhibited both HIV_ADA and HIV_HxB2 pseudovirus infection to a similar degree as T‐20. (g ~ h) SSP‐cell binding assay. GHOST cells were pretreated with SSP or the CCR5 antagonist maraviroc and the CXCR4 antagonist JM2987 as positive controls, and DMSO as a negative control for 1 hr at 37°C prior to being infected with HIV‐1_ADA or HIV‐1_HxB2. SSP pretreatment with the cells had no antiviral effect, whereas maraviroc and JM2987 pre‐treatment showed strong inhibition against HIV‐1_ADA and HIV‐1_HXB2 pseudoviruses at 1 mM, as expected. The data represent the mean ± standard deviation of triplicate experiments

FIGURE 6

Isolation of SSP and UPLC‐MS analysis. (a) UPLC‐DAD spectra of SSP; (b) UPLC spectra of Fr. B (Part I); (c) UPLC spectra of Fr. G (Part II); (d) Extracted ion chromatogram (EIC) of monomer and dimer of the proanthocyanidins in SSP; (e) Extracted ion chromatogram (EIC) of polymers with mDP between 3 and 7 of the proanthocyanidins in SSP; (f) Extracted ion chromatogram (EIC) of polymers with mDP between 8 and 10 of the proanthocyanidins in SSP

FIGURE 7

Antiviral activity of SSP and their extracted parts against SARS‐CoVs, and VSV. As shown in a ~ d, Serially diluted SSP, SSP‐n‐BuOH, Fr. B, F, and G were added to HEK293T‐ACE2 cells infected with SARS‐CoV‐2 (a), SARS‐CoV (b), and VSV (c). The luciferase level was measured 2 or 3 days post‐infection. To test their cytotoxicity, cells viability (d) was also measured using the Promega CellTiter‐Glo Luminescent Cell Viability Assay kit. (E ~ H) Pre‐treatment of SARS‐CoV‐2 and target cells inhibited viral infection. SARS‐CoV‐2 pseudovirus and HEK293T‐ACE2 pre‐treated with serial diluted SSP‐n‐BuOH (e), Fr. B (f), F (g), and G (h) were recovered and subsequently subjected to infect HEK293T‐ACE2, or incubated with untreated SARS‐CoV‐2 pseudovirus for 48 hr, respectively. As controls, HEK293T‐ACE2 cells were infected with SARS‐CoV‐2 pseudovirus and treated with gradient diluted SSP simultaneously. The data represent the mean ± SEM of triplicate experiments.

FIGURE 8

SSP did not show significant cytotoxicity in multiple cell lines, or long‐term in vivo toxicity in rats. (A ~ D) To test SSP cytotoxicity, cells viability was measured in HEK293T‐ACE2 (a), MDCK (b), GHOST (3)‐CD4‐CXCR4 (c) /CCR5 (d). (e) Effects of SSP administration for 4 weeks on the bodyweight of SD rats. (f) Effects of SSP administration for 4 weeks on food intake of SD rats. (g) Effect of SSP administration for 4 weeks on the organ coefficient of SD rats