Inhibition of SARS-CoV 3C-like Protease Activity by Theaflavin-3,3′-digallate (TF3)

SARS-CoV is the causative agent of severe acute respiratory syndrome (SARS). The virally encoded 3C-like protease (3CLPro) has been presumed critical for the viral replication of SARS-CoV in infected host cells. In this study, we screened a natural product library consisting of 720 compounds for inhibitory activity against 3CLPro. Two compounds in the library were found to be inhibitive: tannic acid (IC50 = 3 µM) and 3-isotheaflavin-3-gallate (TF2B) (IC50 = 7 µM). These two compounds belong to a group of natural polyphenols found in tea. We further investigated the 3CLPro-inhibitory activity of extracts from several different types of teas, including green tea, oolong tea, Puer tea and black tea. Our results indicated that extracts from Puer and black tea were more potent than that from green or oolong teas in their inhibitory activities against 3CLPro. Several other known compositions in teas were also evaluated for their activities in inhibiting 3CLPro. We found that caffeine, (—)-epigallocatechin gallte (EGCg), epicatechin (EC), theophylline (TP), catechin (C), epicatechin gallate (ECg) and epigallocatechin (EGC) did not inhibit 3CLPro activity. Only theaflavin-3,3′-digallate (TF3) was found to be a 3CLPro inhibitor. This study has resulted in the identification of new compounds that are effective 3CLPro inhibitors.

developments. In this study, after screening of a natural product library and further confirmation, we found that SARS-3CL Pro could be inhibited by compounds that are abundant in teas. We also examined crude extracts from various teas and a panel of representative natural products in teas for their inhibitory activities against SARS-3CL Pro .

Preparation of SARS-CoV 3CL Pro
Total RNA extracted from a throat swab of a SARS patient was provided by the Center for Disease Control (CDC), Taipei, Taiwan. All RNA samples were handled under bio-safety level 2 (BSL2) regulations. Two primers, 5Ј-GGCGGAATTCGCCTC-TACCAACCACCACAGA-3Ј and 5Ј-GGCGGAATTCA AAG-CATCCAATGATGAGTGCC-3Ј, were used for reverse transcription-polymerase chain reaction (RT-PCR) to synthesize the cDNA of SARS-3CL Pro using Ready-to-Go RT-PCR Beads (Amersham Biosciences, Piscataway, NJ) according to the manufacturer's protocol. The PCR cycle used for the amplification of cDNA of SARS-3CL Pro was as follows: 94ЊC for 5 min, 25 cycles of 94ЊC for 1 min, 58ЊC for 3 min, 72ЊC for 2 min and 72ЊC for 10 min. A full-length 3CL Pro of SARS coronavirus was successfully expressed in Escherichia coli as a glutathione S-transferase (GST) fusion protein. Following cleavage by thrombin, 3CL Pro was purified to homogeneity. A 15mer peptide with sequence H 2 N-SITSAVLQSGFRKMA-COOH corresponding to an autocleavage site of the SARS-3CL Pro was synthesized and used for protease cleavage activity assay. The highly purified protein was shown by high-performance liquid chromatography (HPLC) analysis to exhibit proteolytic activity in trans.

Compound Collection
The Pure Natural Products Library used to screen for 3CL Pro inhibition was obtained from MicroSource Discovery Systems, Inc., Gaylordsville, CT. It consists of a unique collection of 720 pure natural products. The collection includes simple and complex oxygen heterocycles, alkaloids, sequiterpenes, diterpenes, pentacyclic triterpenes, sterols and many other diverse representatives. Initially, 10 different compounds were pooled, and the mixtures were tested for their inhibitory activities against SARS-3CL Pro . Positive pools were then deconvoluted and single pure compounds that were able to inhibit Ͼ50% of peptide cleavage at 10 M were scored as hits. Other compounds were obtained from Sigma (St Louis, MO). Theaflavin (TF1), TF2 [a mixture of TF-2A (theaflavin-3-gallate) and TF-2B (theaflavin-3Ј-gallate)] and theaflavin-3,3Ј-digallate (TF3) were kind gifts from Dr Yu-Chih Liang, School of Medical Technology, Taipei Medical University, Taipei, Taiwan, ROC.

Preparation of Crude Extracts from Oolong Tea, Green Tea, Black Tea and Puer Tea
Tea liquors were prepared from oolong tea, green tea, black tea and Puer tea (also called pu-er or pu-erh) samples as 2% (w/v) tea solutions. The water extracts of four different types of tea leaves were prepared by shaking for 10 min in boiling hot water in thermal flasks. The extracts were then filtered through a Millex-GS 0.22 m filter (Millipore, Malsheim, France) to remove particulate matter. The water extracts were then evaporated under reduced pressure to obtain viscous masses. These materials were evaluated for their inhibitory activities against SARS-3CL Pro .

Inhibition of Protease Activity of SARS-3CL Pro by HPLC Assay
A general cysteine protease inhibitor, N-ethylmaleimide (NEM, protease inhibitor as positive control), was obtained from Sigma (St Louis, MO). This compound was tested for its inhibitory activities against SARS-3CL Pro in the peptide cleavage assay. The activities of the proteases were measured in the absence or presence of different concentrations of these general inhibitors. The enzyme (2 M) was pre-incubated with chemicals for 30 min. Peptide was then added at 100 M to the reaction, and mixtures were incubated at 37ЊC for 1 h. The potency of the inhibitors was calculated. The HPLC analysis condition is: a C 18 RP guard column (250 ϫ 4.6 mm ϫ 5 m, Agilent Zorbax Extend). This method was chosen because it gave the best resolution among several methods that were tested. The column temperature was ambient, and the elution (0.8 ml/min) was performed using a solvent system comprising solvents A [10 MM NH 4 OAc, 0.1% trifluoroacetic acid (TFA)] and B (acetonitrile, 0.1% TFA) mixed using a gradient starting with 100% A, linearly decreasing to 10% A in 8 min, to 100% A in 16 min, and held at 100% A for 23 min. The injection volume was 40 l. The UV detector was set at 214 nm.

Proteolytic Activity of SARS-3CL Pro by Fluorogenic Substrate Peptide Assay
The development of a fluorescent protease activity assay has been reported previously (7). The enhanced fluorescence due to cleavage of the fluorogenic substrate peptide (Dabcyl-KTSAVLQSGFRKME-Edans) catalyzed by the protease was monitored at 538 nm with excitation at 355 nm using a fluorescence plate reader (Fluoroskan Ascent from ThermoLabsystems, Sweden). The fluorimetric assay was used to determine the IC 50 of identified inhibitors on SARS 3CL Pro activity. The protease was stored in the buffer containing 12 mM Tris-HCl (pH 7.5), 120 mM NaCl, 0.1 mM EDTA, 7.5 mM 2-mercaptoethanol and 1 mM dithiothreitol (DTT) at Ϫ70ЊC before use.

Screening of a 720 Compound Library Against 3CL Pro Activity by HPLC Assay
The spectrum collection, a library of 720 drugs and natural products, was screened for 3CL Pro inhibitory activity. A flowchart of the screening procedure is shown in Fig. 1. Initially, 10 different compounds were pooled, and the 72 mixtures were then tested for their inhibitory activities against SARS-3CL Pro . The HPLC assay was used in the primary screening because many compounds in the library are chromogenic. Single compounds in the positive pools were then evaluated for their activities. Two compounds, tannic acid and TF2B, were found to be active against 3CL Pro (Fig. 2). Tannic acid is a water-soluble polyphenol present in plant food such as tea, red wine, beer and nuts. In addition, tea polyphenols and many tannin components are thought to be anticarcinogenic (14,15).   Figure 2. Inhibition of 3CL Pro activity by HPLC assay. Injection of 40 l of incubation samples for C18 reverse phase column analysis. The substrate or product fragment peaks were analyzed by HPLC with UV absorbance detection at 214 nm. The HPLC system is described in Methods.

Evaluation of Tea Extracts and Pure Components from Teas for Their Inhibition of 3CL Pro Activity
Since many other polyphenols related to tannic acid and TF2B are also present in various kinds of teas, we decided to examine the inhibition of activity by various tea extracts and several well known pure ingredients present in teas. Thus, water extracts from different types of teas were prepared and evaluated for their inhibitory activities against 3CL Pro . As shown in Table 1, extracts from black and Puer teas inhibited 3CL Pro activity while those from green or oolong teas did not. We further tested the possible involvement of several known ingredients present in teas, including caffeine, theophylline, catechin (C), epigallocatechin (EGC), (Ϫ)-epigallocatechin gallte (EGCg), epicatechin (EC), epicatechin gallate (ECg), TF1, TF2, TF2B, tannic acid and TF3. As shown in Table 1, the results showed that methylxanthine (caffeine and theophylline) did not influence 3CL Pro activity; catechins, including C, EC, ECg, EGC and EGCg, present in green tea (unfermented) and oolong tea (partial fermented) also did not inhibit 3CL Pro activity. Our results indicated that tannic acid, TF2B and TF3 are 3CL Pro inhibitors as revealed by the fluorogenic substrate assay (Fig. 3). TF2A was not tested because of its unavailability.

TF2B and TF3 as Potent Agents Against 3CL Pro Inhibitory Activity
The natural product screened library consisted of simple and complex oxygen heterocycles, alkaloids, sequiterpenes, diterpenes, pentacyclic triterpenes, sterols and many other diverse representatives. Numerous black tea polyphenols (especially those of TF1, TF2 and TF3) were found to inhibit 3CL Pro . In black tea, TF3 was the most abundant (1.05%) followed by TF2A (0.34%), TF2B (0.11%) and TF1 (0.08%) (16). It is interesting to note that TF3 contained two gallate groups attached to the 3,3Ј positions as shown in Fig. 4b. TF2A and

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Inhibition of SARS-CoV 3C-like protease activity Relative 3CL pro activity (%) Figure 3. Inhibition of 3CL Pro activity by fluorogenic substrate peptide assay. Inhibition of 3CL Pro activity by tannic acid, TF1, TF2B, TF3 and EGCg. The enhanced fluorescence due to cleavage of the fluorogenic substrate peptide catalyzed by the protease was monitored at 538 nm with exicitation at 355 nm using a fluorescence plate reader. The determining assay is described in Methods. TF2B, on the other hand, consisted of only one gallate group attached either to the 3 or the 3Ј positions, respectively (Fig. 4b). In contrast, no gallate group was attached to TF1. Results in Table 1, indicating that the gallate group attached to the 3Ј position to TF2B and TF3, might be important for interaction with the 3CL Pro active site. EC, EGCg, ECG and EGC were much less active (IC 50 Ն 100 M) than theaflavins.

Discussion
Human coronaviruses (HCoVs) are major causes of upper respiratory tract illness in animals including humans. It has been demonstrated that a novel coronavirus causes SARS. The main protease, frequently called 3C-like protease (3CL Pro ), has been identified as an attractive drug target (17). Of the 720 natural products screened in this study, tannic acid (Fig. 4a) and TF2B were identified using the HPLC proteolytic assay to inhibit 50% of the proteolytic acitivity of 3CL Pro at concentrations Ͻ10 M. Both tannic acid and TF2B belong to the group of natural polyphenols found in teas. Low concentrations of tannic acid were found to inhibit proteases including tissue-type plasminogen activator, urokinase-type plasminogen activator and plasmin activity [13]. Over 300 different kinds of tea are now produced, but they are classified into three main forms: green tea is manufactured by drying fresh tea leaves, with emphasis on the prevention of oxidation of the tea polyphenols during the manufacturing process. In contrast, the manufacture of black tea is characterized by a high degree of fermentation, which produces a series of chemical condensations among the ingredients present in tea. Oolong tea is partially fermented, and the tea compositions are more similar to those found in green tea (18). In evaluating different types of tea, we found that extracts from several different types of tea, including Puer and black tea, could inhibit 3CL Pro activity while green or oolong tea could not. However, the chemical composition in Puer tea is very complex and it is difficult to isolate pure ingredients for structural identification.
Purification and identification of Puer tea ingredients should be carried out in a separate study.
We next examined whether other well-known ingredients present in tea could also inhibit 3CL Pro activity. We found that methylxanthine (caffeine and theophylline) and catechins (EGCg, EC, C, ECg and EGC) were not able to inhibit 3CL Pro activity at concentrations up to 100 M (Table 1). However, TF1, TF2 and TF3 were more potent 3CL Pro inhibitors than catechins in green tea. During the fermentation step in the production of black tea, most of the catechins are oxidized and condensed into theaflavins through dimerization and into thearubigins through polymerization. Green tea contains ϳ30% of catechins (dry mass base) while black tea contains ϳ9% of catechins and 4% of theaflavins (19). In another study, Leung et al. (16) reported that TF3 was the most abundant (1.05%) theaflavin in black tea followed by TF2A (0.34%), TF2B (0.11%) and TF1 (0.08%). Tannic acid is a class of polyphenolic in plants but the quantification of its levels in green or black tea is difficult because tannic acid constitutes a wide range of polymers with mol. wts of 500-3000 Da.
It is interesting to note that TF2B and TF3 are more potent inhibitors of 3CL Pro than TF1 (Table 1). Unlike TF2B and TF3, TF1 does not contain a gallate group (Fig. 4b). Thus, the addition of a gallate group attached at the 3Ј position to TF2B and TF3 might be important for their inhibitory activity against 3CL Pro . These results suggest that TF2B and TF3 might be good starting points for the design of more active inhibitors for the 3CL Pro encoded by SARS-CoV.
Finally, this study has identified three compounds (TF2B, TF3 and tannic acid) that are effective 3CL Pro inhibitors (IC 50 Ͻ10 M). These compounds are abundant in the extract of black tea (16,19). Black tea is a popular beverage in the world. Results from this study warrant further investigation to examine the effect of these natural products in inhibition of SARS-CoV replication in cell culture. Clark et al. reported that theaflavins extracted from black tea were able to neutralize bovine coronavirus and rotavirus infections (20). Thus, it will be very interesting to evaluate, in a separate study, whether drinking black tea can prevent or alleviate the infection of an enteric form of coronavirus since SARS-CoV is known to actively replicate in the intestinal tract (21).