Inhibition of SARS Coronavirus Helicase by Baicalein

Abstract

The severe acute respiratory syndrome (SARS) occurred in the southern part of China in the late 2002 and rapidly spread to Canada and Southeast Asian countries, including Singapore, Vietnam, Hong Kong, and Taiwan. During the outbreak, clinicians were unaware of which treatments were appropriate to treat SARS patients because they had no prior experience in dealing with this disease. Therefore, they prescribed the SARS patients with general antiviral medications, such as ribavirin, type-I interferon and corticosteroids. The SARS pandemic was finally controlled within a year of outbreak through progressive global efforts, including patient quarantine. A retrospective case-controlled analysis showed that these antiviral agents had minimal beneficial effects or even worsened the symptoms of SARS patients, suggesting that it is necessary to develop effective anti-SARS agents for another SARS outbreak in the future. A novel coronavirus (CoV) was identified as a causal factor for the SARS. Coronaviruses are members of a family of enveloped viruses that replicate in the cytoplasm of host cells. Analysis of the full sequence of the SARS coronavirus (SCV) revealed that it is a large, single-stranded, and positive-sense RNA virus, which bears a moderate resemblance with other human coronaviruses, HCoV-OC43 and HCoV229E. Notably, the first 2/3 of the SCV genome consists of the viral replicase genes that encode 16 non-structural proteins (nsPs), including the NTPase/helicase (nsP13). The SCV helicase, nsP13 is a critical component for viral replication and currently regarded as a feasible drug target for potential SCV chemical inhibitors. Helicase is a molecular motor protein that separates double-stranded nucleic acid (NA), using the free energy generated from nucleoside triphosphate (NTP) hydrolysis during translocation on singlestranded NA. The nsP13 is considered as a RNA helicase because SCV is a positive-strand ssRNA virus and comprise three major domains: Zn ion binding domain, hinge domain and NTPase/helicase domain. Previous studies have identified numerous natural or synthetic chemicals that suppress the nsP13 activity with different modes of actions. For example, Tanner et al. have demonstrated that the bananin derivatives are non-competitive inhibitors of the ATPase activity of nsP13. Yang et al. have demonstrated that bismuth complexes compete for the Zn ion binding sites, thereby exhibiting significant inhibitory effects on both the ATPase and DNA duplex-unwinding activities of nsP13. Lee et al. have found that aryl diketoacid (ADK) analogs suppress the nsP13 activity. Because ADKs are well-known metal chelators, authors initially speculated that ADKs would inhibit the activity of nsP13 in an analogous manner to the bismuth complexes: ADKs bind to Zn ion binding site, thereby inhibiting both the ATPase and DNA duplex-unwinding activities. Contrary to their expectations, however, they observed that ADKs selectively inhibited the duplex DNA-unwinding activity, but not the ATPase activity of nsP13 and the anti-SCV helicase activities of ADKs were dependent on regiochemistry as well as the substituent of ADKs, leading to the conclusion that alternative inhibitory mechanism(s) of the nsP13 activity by ADKs might exist. In accordance with the above observations, we have recently conducted in vitro biochemical experiments to find out dietary or medicinal compounds that possess significant inhibitory effects on the nsP13 helicase activity. As a result, we found that both myricetin and scutellarein (Fig. 1) strongly inhibited the ATPase activity, but not the DNA unwinding activity of nsP13. IC50 values of myricetin and scutellarein against the ATPase activity of nsP13 were observed to be 2.71 ± 0.19 μM and 0.86 ± 0.48 μM, respectively. It is notable that myricetin and scutellarein are natural flavonoids. Flavonoids are secondary plant metabolites that