Polymethoxyflavone from Citrus depressa as an inhibitor against various variants of SARS-CoV-2 spike protein

In traditional Taiwanese medicine, Citrus depressa Hayata serves as the raw material of Chen-Pi which has been widely used to treat respiratory ailments. Scientific investigations have validated the attributes of C. depressa, elucidating its valuable properties, including antioxidative, anti-inflammatory, anticancer, neuroprotion, hepatoprotection, and hypolipidemic effects.

This study aims to isolate a universal inhibitor of the SARS-CoV-2 spike protein from C. depressa and confirm the mechanism by which these inhibitors disrupt the binding of the spike protein to hACE2.

The whole fruit of C. depressa was subjected to ethanol extraction, following by partitioning to obtain water, butanol, and ethyl acetate fractions. To identify the inhibitory components in citrus fruits, we performed both the SPR assay and the SARS-CoV-2 pseudo-virus assays. Subsequently, we employed a bioassay-guided approach to efficiently isolate and characterize the bioactive constituents that hindered the interaction between the SARS-CoV-2 spike protein and hACE2, using a combination of MPLC and Semi-preparative HPLC for compound isolation. ELISA based spike protein binding assay evaluate the inhibitory activities of the extract and potential constituents against multiple spike protein variants. To further shed light on the inhibitory mechanism, candidate inhibitors were validated through the SPR assay and molecular docking.

The crude extract and ethyl acetate layer derived from C. depressa showed significant inhibitory activity on SARS-CoV-2 Omicron BA.4/5, with IC₅₀ of 77.4 μg/mL and 100 μg/mL, respectively. Ten potential compounds from C. depressa have been identified with inhibitory activity against various SARS-CoV-2 spike proteins. 2'-hydroxy-4,4',5',6'-tetramethoxychalcone (Cd3) and 5-hydroxy-3',4',6,7,8-pentamethoxyflavone (Cd8) also showed good inhibitory activity to the spike protein, with K_D of 0.79 μM and 37.3 nM, respectively. These findings are in line with prior study, indicating Cd3 and Cd8 can bind to key amino acid residue, disrupting the formation of the spike protein and h-ACE2 complex.

This study presents the initial evidence showcasing the inhibitory effect of polymethoxyflavones (PMFs) on the spike protein of SARS-CoV-2. Moreover, the inhibitory activity of C. depressa extracts indicates their potential to prevent infections of different SARS-CoV-2 variants.