Potential antiviral activity of isorhamnetin against SARS-CoV-2 spike pseudotyped virus in vitro

Abstract

Coronavirus Disease 2019 (COVID-19) cases and deaths are still rising worldwide, there is currently no effective treatment for severe inflammation and acute lung injury caused by new coronavirus (SARS-COV-2) infection. Therapies to prevent or treat COVID-19, including antiviral drug and several vaccines, are still being development. Human angiotensin-converting enzyme 2 (ACE2), expressing in lung, has been confirmed to be a receptor for SARS-COV-2 infection, interventions for attachment of spike protein of SARS-CoV-2 to ACE2 may be a potential approach to prevent viral infections and it is considered as a potential target for drug development. In this study, we observed that seabuckthorn and its flavonoid compounds quercetin and isorhamnetin were shown strong retention to ACE2 overexpression HEK293 (ACE2^h ) cells by CMC analysis. Based on drug receptor interaction analysis and viral entry studies in vitro, we evaluated the interaction of two flavonoid compounds and ACE2 as well as the inhibitory effect of the two compounds on viral entry. Surface plasmon resonance assay proved the effect that isorhamnetin bound to the ACE2, and its affinity (KD value) was at the micromolar level, that was, 2.51 ± 0.68 μM. Viral entry studies in vitro indicated that isorhamnetin inhibited SARS-CoV-2 spike pseudotyped virus entering ACE2^h cells. Based on promising in vitro results, we proposed isorhamnetin to be a potential therapeutic candidate compound against COVID-19.

Keywords: SARS-CoV-2; antiviral activity; isorhamnetin.

FIGURE 1

The chromatograms of sea‐buckthorn extract (SBE), quercetin and isorhamnetin on ACE2/HEK293/CMC affinity column

FIGURE 2

Determination of the equilibrium dissociation constant (KD) of quercetin and isorhamnetin with ACE2 by SPR analysis

FIGURE 3

Evaluation of the toxicity of quercetin and isorhamnetin on ACE2^h cells by the change of cell viability and intracellular Ca²⁺ flux. (a and b) Effect of quercetin (a) and isorhamnetin (b) on the viability of ACE2^h cells. ACE2^h cells were treated with different concentrations of quercetin and isorhamnetin for 24 h, cells viability was tested by CCK8 assay. (c and d) Effect of quercetin (c) and isorhamnetin (d) on intracellular Ca²⁺ flux change in ACE2^h cells. ACE2^h cells were pretreated with 100 μM concentrations of quercetin and isorhamnetin, intracellular Ca²⁺ flux was tested by Ca²⁺ mobilization assay. The experiments were repeated three times. Data are presented as mean ± SEM

FIGURE 4

Effect of quercetin and isorhamnetin on the entrance of 2019‐nCoV spike pseudotyped virus into ACE2^h cells. ACE^h cells infected with 2019‐nCoV spike pseudotyped virus were considered as control, which luciferase luminescence value was defined as 1. The experiments were repeated three times. Data are presented as mean ± SEM. **p < .01 compared with control

FIGURE 5

Molecular docking results of isorhamnetin