key: cord-0732217-x0z68jos
authors: Lin, Cheng-Wen; Tsai, Fuu-Jen; Tsai, Chang-Hai; Lai, Chien-Chen; Wan, Lei; Ho, Tin-Yun; Hsieh, Chang-Chi; Chao, Pei-Dawn Lee
title: Anti-SARS coronavirus 3C-like protease effects of Isatis indigotica root and plant-derived phenolic compounds
date: 2005-08-09
journal: Antiviral Res
DOI: 10.1016/j.antiviral.2005.07.002
sha: fec5f908de12a2d95ef4c09ad412b1ae2f0591b0
doc_id: 732217
cord_uid: x0z68jos

The 3C-like protease (3CL(pro)) of SARS-coronavirus mediates the proteolytic processing of replicase polypeptides 1a and 1ab into functional proteins, becoming an important target for the drug development. In this study, Isatis indigotica root extract, five major compounds of I. indigotica root, and seven plant-derived phenolic compounds were tested for anti-SARS-CoV 3CL(pro) effects using cell-free and cell-based cleavage assays. Cleavage assays with the 3CL(pro) demonstrated that IC(50) values were in micromolar ranges for I. indigotica root extract, indigo, sinigrin, aloe emodin and hesperetin. Sinigrin (IC(50): 217 μM) was more efficient in blocking the cleavage processing of the 3CL(pro) than indigo (IC(50): 752 μM) and beta-sitosterol (IC(50): 1210 μM) in the cell-based assay. Only two phenolic compounds aloe emodin and hesperetin dose-dependently inhibited cleavage activity of the 3CL(pro), in which the IC(50) was 366 μM for aloe emodin and 8.3 μM for hesperetin in the cell-based assay.

Severe acute respiratory syndrome (SARS) was reported in 8447 cases with 811 deaths worldwide from February to June 2003 (Poutanen et al., 2003; Peiris et al., 2003; Drosten et al., 2003) . A novel coronavirus, SARScoronavirus (SARS-CoV) was identified as the etiological agent of the disease (Ksiazek et al., 2003; Lee et al., 2003; Tsang et al., 2003; Hsueh et al., 2003) . SARS-CoV particle contains a single positive-stranded RNA genome encoding for replicase, spike, envelope, membrane, and nucleocapsid (Lai, 2003; Enjuanes et al., 2001; Holmes, 2003) . The SARS-CoV 3CL pro mediates the proteolytic processing of replicase polypeptides into functional proteins, playing an important role in viral replication. Therefore, the SARS-CoV 3CL pro can be considered an attractive target for developing effective drugs against SARS. Several potential 3CL pro inhibitors with a 50% inhibitory concentration (IC 50 ) below 10 M were identified from the large number of the structurally diverse small molecules (Kao et al., 2004; Hsu et al., 2004) .

Isatis indigotica root and phenolic Chinese herbs were frequently used for the prevention of SARS during the SARS outbreaks in China, Hong Kong, and Taiwan. I. indigotica root (Radix isatidis), belonging to the family Cruciferae, is native to China. Antiviral effects of I. indigotica root were found against influenza, hepatitis A and Japanese encephalitis (Qin and Xu, 1998; Wu et al., 1997) . I. indigotica root contains indigo, indirubin, indican (indoxyl-␤-d-glucoside), ␤-sitosterol, ␥-sitosterol, sinigrin, etc. (Gilbert et al., 2004) . Indigo and indirubin were identified as the promiscuous chymotrypsin inhibitors (McGovern and Shoichet, 2003 Recently, an anti-influenza virus effect of indirubin has been demonstrated (Mak et al., 2004) . In addition, several herbderived phenolics aloeemodin, hesperetin, quercetin, and naringenin have been accredited with antiviral effects against poliovirus, vesicular stomatitis virus, Sindbis virus, herpes simplex virus types 1 and 2, parainfluenza virus, and vaccinia virus (Semple et al., 2001; Andersen et al., 1991; Paredes et al., 2003; Kim et al., 2001) .

In this study, we characterized the anti-SARS-CoV 3CL pro effect of the water extract of I. indigotica root, I. indigotica root-derived compounds, and herb-derived phenolics using a cell-free cleavage and cell-based cleavage assay.

The root of I. indigotica was purchased from Sun Ten Pharmaceutical Corporation (Taiwan). The plant root of Isatidis indigotica was extracted twice with 10 volumes of distilled boiling water for 1 h. The aqueous extract was concentrated under the reduced pressure at 50 • C, passed through 0.22-m filters for sterilization, and diluted in culture medium to make a stock concentration of 10 mg/ml. Indigo and indirubin were kindly provided by Dr. Yuan-Shiun Chang, professor for Institute of Chinese Pharmaceutical Sciences, China Medical University. Indican (indoxyl-␤-dglucoside), ␤-sitosterol, sinigrin, aloe emodin, hesperetin, quercetin, naringenin, daidzein, emodin, and chrysophanol were purchased from Sigma Chemical.

To examine the trans-cleavage of SARS-CoV 3CL pro in the cell-free assay, recombinant 3CL pro was expressed in E. coli and purified using the HisTrap Kit (Amersham) as described in our previous report . Coomassie Blue-staining revealed that recombinant 3CL pro contained a major 34-kDa band for the monomer and a minor 68-kDa band for the dimer (Fig. 1A, lane 2) . The cleavage substrate (TVRLQAGNATE) was generated as the substrate fusion protein with the N-terminal S-Tag and the C-terminal HSV-Tag. In the cell-free cleavage assay, the substrate fusion protein that was captured by anti-HSV-Tag antibodies in wells incubated with soluble 3CL pro for 3 h at 37 • C. The non-cleavage substrate protein was detected by an Enzyme Linked Immunosorbent Assay (ELISA) using peroxidaseconjugated S protein. ELISA showed that cell-free proteolytic activity correlated, in concentration-dependent manner, with the serial twofold dilution of recombinant 3CL pro protein in the range from 15 g/ml to 240 g/ml (Fig. 1B) . Subsequently, the anti-3CL pro effect by the extract of I. indigotica root was evaluated using the cell-free cleavage assay. The cell-free cleavage assay indicated that the extract of I. indigotica root had a dose-dependent anti-3CL pro effect with an IC 50 of 53.8 ± 4.2 g/ml ( Fig. 2 ; Table 1 ).

The cell-based cleavage assay of 3CL pro for screening inhibitors does not require purification of the active 3CL pro , The trans-cleavage of the 3CL pro with a substrate fusion protein was determined using the ELISA. The substrate fusion protein was captured with anti-HSV mAb, followed by incubation with the serial dilution of the 3CL pro . The non-cleavage of the fusion protein was detected using the S protein-HRP conjugate and ABTS/H 2 O 2 substrates. The ELISA product was measured at A405 nm. The relative cell-free cleavage activity was calculated as 1 − (A405 3CL pro )/(A405 no 3CL pro ). Table 1 The inhibitory effect on cell-free and cell-based cleavage activity of the SARS-CoV 3CL pro and represents closely the natural physiological state. Therefore, we used the cell-based cleavage assay for examining the inhibitory efficacy of the 3CL pro inhibitors. For the cellbased cleavage assay, the in-frame construction of the 3CL pro , the substrate, and the luciferase, designed as the plasmid pcDNA3.1-3CL pro -S-Luc, was co-transfected with the indicated vector pEGFP-N1 into Vero cells. The stable cell clone for the expression of the 3CL pro -substrate-luciferase fusion protein was selected by Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS and 800 g/ml of neomycin G418 (Fig. 3A) . Since a more than 30 kDa protein fused at the N-terminus of the luciferase resulted in a dramatic decrease of luciferase activity (Joubert et al., 2000) , the detection of luciferase activity could be considered as a measure for the cis-cleavage by the SARS-CoV 3CL pro . Western blotting with the anti-luciferase monoclonal antibody showed a 94-kDa band for the fusion protein 3CL pro -S-Luc and a 60-kDa band for the luciferase in Vero cells transfected with the plasmid pcDNA3.1-3CL pro -S-Luc (data not shown). The relative luciferase activity in the transfected cells was subsequently measured using the dual Luciferase Reporter Assay System (Fig. 3B ). In the cell-based cleavage assay, the extract of I. indigotica root significantly inhibited the cis-cleavage activity of the SARS-CoV 3CL pro with an IC 50 of 191.6 ± 8.2 g/ml ( Fig. 4 ; Table 1 ). The IC 50 value from cell-based assay by the I. indigotica root extract was twofold higher than the IC 50 value from the cell-free assay. The reason may be that the I. indigotica root extract could contain some inhibitory compounds that cannot permeate cellular membranes to reach intracellular SARS-CoV 3CL pro . The results of cell-free and cell-based cleavage assays demonstrated that the I. indigotica root extract might contain potent anti-SARS-CoV 3CL pro compounds. The in vitro cytotoxicity profile of the I. indigotica root extract was examined using Vero cells. Vero cells in MEM medium with 10% FBS were plated in 96-well plates (5 × 10 4 cells/well) and then treated with the indicated compounds. After the treatment for 20 h, 25 l of a MTT solution at 5 mg/ml was added to each well and incubated at 37 • C in 5% CO 2 for 3 h. After a three-time washing of phosphate buffer saline, 100 l DMSO was then added into the plates for dissolving the formazan crystals. OD 570-630 in each well was then measured with a micro-ELISA reader. The result indicated that the 50% cytotoxic concentration (CC 50 ) was greater than 5000 g/ml.

Five major compounds of the I. indigotica root, including indigo, indirubin, indican, sinigrin, and beta-sitosterol were further tested for anti-SARS-CoV 3CL pro action (Fig. 5 , Table 1 ). Of the five compounds, sinigrin, beta-sitosterol and indigo dose-dependently inhibited cleavage activities of the 3CL pro in cell-free and cell-based assays (Fig. 5 , Table 1 ). The IC 50 in the cell-free assays was 115 M for beta-sitosterol, 121 M for sinigrin, and 300 M for indigo. The cell-based assay indicated that sinigrin (IC 50 : 217 M) was more efficient in blocking the cleavage processing of the 3CL pro than indigo (IC 50 : 752 M) and beta-sitosterol (IC 50 : Fig. 5 . Inhibition of the cell-free cleavage of the 3CL pro by the compounds derived from Isatis indigotica root. The indicated compound was added into the mixture of the substrate fusion protein and the 3CL pro , and then the uncleaved substrate was detected using the S protein-HRP conjugate and ABTS/H 2 O 2 substrates. The ELISA product was measured at A405 nm. The relative inhibition of cell-free cleavage activity was calculated as 1 − (A405 no 3CL pro − A405 3CL pro with inhibitor )/(A405 no 3CL pro − A405 3CL pro ).

1210 M). Sinigrin showed a strong correlation between the effects on cell-free and cell-based cleavage of the SARS-CoV 3CL pro . Moreover, indigo (CC 50 : 7.4 mM) and sinigrin (CC 50 : >10 mM) were not toxic to Vero cells. Sinigrin, an antioxidant, has been reported to possess inhibitory effects on quinine reductase and glutathione S-transferase, antiproliferative effects against cancer cells, and antimicrobial activity against Bacillus subtilis and Saccharomyces cerevisiae (Brabban and Edwards, 1995; Munday and Munday, 2002; Smith et al., 2004) . This study is the first report in that sinigrin significantly blocks the cleavage processing of a viral protease.

Seven phenolic compounds, aloeemodin, hesperetin, quercetin, naringenin, daidzein, emodin, and chrysophanol were also tested for their inhibitory effects on the SARS-CoV 3CL pro ( Fig. 6; Table 1 ). Only two of the phenolic compounds, aloeemodin and hesperetin dose-dependently inhibited cleavage activity of the 3CL pro in cell-free and cell-based assays ( Fig. 6; Table 1 ). In the cell-free assay, the IC 50 values were 132 M for aloe emodin and 60 M for hesperetin. Quercetin has been reported to block the entry of SARS-CoV into host cells (Yi et al., 2004) . However, no inhibitory effect on SARS-CoV 3CL pro was found for quercetin in the cell-free and cell-based cleavage assays. Interestingly, hesperetin (CC 50 : 2.7 mM) had an IC 50 of 8.3 M in the cell-based assay (Table 1) . Hesperetin is poorly soluble in water; so, hesperetin was less inhibitory in the cell-free assay than in the cell-based assay. The finding of the anti-3CL pro effects of hesperetin at the micromolar range was consistent with a previous report indicating that hesperetin had an inhibitory activity on Sindbis virus infection with an IC 50 of 20.5 g/ml (about 68 M) by plaque assay (Paredes Fig. 6 . Inhibition of the cell-free cleavage of the 3CL pro by the phenolic compounds. The indicated compound was added into the mixture of the substrate fusion protein and the 3CL pro , and then the uncleaved substrate was detected. The relative inhibition of cell-free cleavage activity was calculated as 1 − (A405 no 3CL pro − A405 3CL pro with inhibitor )/(A405 no 3CL pro − A405 3CL pro ). et al., 2003) . Of the compounds tested, hesperetin was the most potent inhibitor of SARS-CoV 3CL pro (Table 1) .

Our results have demonstrated significantly inhibitory effects on SARS-CoV 3CL pro by I. indigotica root extract, indigo, sinigrin, aloeemodin and hesperetin in the micromolar range. Particularly, the cell-based assay demonstrated that hesperetin (IC 50 : 8.3 M) and sinigrin (IC 50 : 217 M) could be potential inhibitors of SARS-CoV 3CL pro . In addition, sinigrin and hesperetin with a CC 50 of over 2 mM were considerably less cytotoxic to Vero cells (Table 1 ). Akin to other reported anti-SARS substances, such as glycyrrhizin (Cinatl et al., 2003a) , nelfinavir (Yamamoto et al., 2004) , aurintricarboxylic acid , and interferon (Cinatl et al., 2003b) , the compounds reported here may be considered as potential leads in the development of inhibitors of SARS-CoV 3CL pro .

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We would like to thank the National Science Council (Taiwan) and China Medical University for financial support (NSC93-2320-B-039-051, NSC 92-2314-B-039-030, NSC 92-2751-B-039-009-Y, and CMU92-MT-03).