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Severe acute respiratory syndrome (SARS) is an infectious disease with a strong potential for transmission upon close personal contact and is caused by the SARS-coronavirus (CoV). However, there are no natural or synthetic compounds currently available that can inhibit SARS-CoV. We examined the inhibitory effects of 64 purified natural compounds against the activity of SARS helicase, nsP13, and the hepatitis C virus (HCV) helicase, NS3h, by conducting fluorescence resonance energy transfer (FRET)-based double-strand (ds) DNA unwinding assay or by using a colorimetry-based ATP hydrolysis assay. While none of the compounds, examined in our study inhibited the DNA unwinding activity or ATPase activity of human HCV helicase protein, we found that myricetin and scutellarein potently inhibit the SARS-CoV helicase protein in vitro by affecting the ATPase activity, but not the unwinding activity, nsP13. In addition, we observed that myricetin and scutellarein did not exhibit cytotoxicity against normal breast epithelial MCF10A cells. Our study demonstrates for the first time that selected naturally-occurring flavonoids, including myricetin and scultellarein might serve as SARS-CoV chemical inhibitors.

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Exposure of sulforaphane to HepG2 cells increased heme oxygenase-1 (HO-1) expression by activating antioxidant response element (ARE) through induction of Nrf2 and suppression of Kelch-like ECH-associated protein 1 (Keap1). Using human HO-1 promoter reporter plasmids and ChIP assay, we have identified that sulforaphane transcriptionally activated the upstream ARE-rich enhancer region, located at À9.0 kb upstream human HO-1 promoter. Induction of HO-1 by sulforaphane was attenuated by overexpression of mutant Nrf2 plasmid in HepG2 cells and totally abolished in Nrf2 knockout mouse embryonic keratinocytes and fibroblasts. Overexpression of individual p38 mitogen-activated protein (MAP) kinase (MAPK) isoforms also suppressed constitutive as well as sulforaphane-or Nrf2-induced ARE-dependent gene expression. Among the upstream kinases, although MKK3 was not involved in suppression of ARE by any of p38 MAPK isoforms, MKK6 selectively suppressed ARE by p38; or p38D, but not by p38A or p38B. Importantly, sulforaphane not only activated MAP/extracellular signal-regulated kinase (ERK) kinases 1/2 and ERK1/2, but also strongly suppressed anisomycin-induced activation of p38 MAPK isoforms by blocking phosphorylation of upstream kinases, MKK3/6. Finally, we found that stimulation of p38 MAPK isoforms phosphorylated purified Nrf2 protein and caused an increase in the interaction between Nrf2 and Keap1 in vitro and the suppression of Nrf2 translocation into the nucleus. Collectively, our results indicate that transcriptional activation of Nrf2/ ARE is critical in sulforaphane-mediated induction of HO-1, which can be modulated in part by the blockade of p38 MAPK signaling pathway. In addition, our study shows that p38 MAPK can phosphorylate Nrf2 and promotes the association between Nrf2 and Keap1 proteins, thereby potentially inhibiting nuclear translocation of Nrf2. (Cancer Res 2006; 66(17): 8804-13)