key: cord-321714-yhruzu7f
authors: Ryu, Young Bae; Park, Su-Jin; Kim, Young Min; Lee, Ju-Yeon; Seo, Woo Duck; Chang, Jong Sun; Park, Ki Hun; Rho, Mun-Chual; Lee, Woo Song
title: SARS-CoV 3CL(pro) inhibitory effects of quinone-methide triterpenes from Tripterygium regelii
date: 2010-03-15
journal: Bioorg Med Chem Lett
DOI: 10.1016/j.bmcl.2010.01.152
sha: 
doc_id: 321714
cord_uid: yhruzu7f

Quinone-methide triterpenes, celastrol (1), pristimerin (2), tingenone (3), and iguesterin (4) were isolated from Triterygium regelii and dihydrocelastrol (5) was synthesized by hydrogenation under palladium catalyst. Isolated quinone-methide triterpenes (1–4) and 5 were evaluated for SARS-CoV 3CL(pro) inhibitory activities and showed potent inhibitory activities with IC(50) values of 10.3, 5.5, 9.9, and 2.6 μM, respectively, whereas the corresponding 5 having phenol moiety was observed in low activity (IC(50) = 21.7 μM). As a result, quinone-methide moiety in A-ring and more hydrophobic E-ring assist to exhibit potent activity. Also, all quinone-methide triterpenes 1–4 have proven to be competitive by the kinetic analysis.

Quinone-methide triterpenes, celastrol (1), pristimerin (2), tingenone (3) , and iguesterin (4) were isolated from Triterygium regelii and dihydrocelastrol (5) was synthesized by hydrogenation under palladium catalyst. Isolated quinone-methide triterpenes (1-4) and 5 were evaluated for SARS-CoV 3CL pro inhibitory activities and showed potent inhibitory activities with IC 50 values of 10.3, 5.5, 9.9 , and 2.6 lM, respectively, whereas the corresponding 5 having phenol moiety was observed in low activity (IC 50 = 21.7 lM).

As a result, quinone-methide moiety in A-ring and more hydrophobic E-ring assist to exhibit potent activity. Also, all quinone-methide triterpenes 1-4 have proven to be competitive by the kinetic analysis.

Ó 2010 Elsevier Ltd. All rights reserved.

The worldwide outbreak of the life-threatening disease severe acute respiratory syndrome (SARS) was caused by infection with SARS-CoV as a novel coronavirus (CoV). 1, 2 The genome of SARS-CoV contains 11-14 major annotated open reading frames including those predicted to encode for known viral proteins, including the replicase polyproteins, S (spiked protein), polymerase, M (membrane protein), N (nucleocapsid protein), and E (small envelope protein). 2 The replicase polyproteins ppla and pplb encoded by the SARS-CoV are extensively processed to yield the functional subunits for successful viral propagation. 3 The SARS-CoV 3CL pro , which is also called main protease (M pro ) mediates the proteolytic processing of replicase polypeptides into functional proteins, plays an important role in viral replication. The active site of SARS-CoV 3CL pro has a catalytic dyad with the sulfur of Cys145 as a nucleophile and the imidazole ring of His41 as a general base. 4 Therefore, the SARS-CoV 3CL pro can be considered an attractive target for developing effective drugs against SARS.

To date, a large number of inhibitors of 3CL pro have been studies, including C2-symmetric diols, 5 isatin derivatives, 6 anilides, 7 and thiazolyl ketone-containing peptidic compounds. 8 Moreover, some groups have reported that triterpenoids such as betulinic acid from Juniperus formosana 9 and glycyrrhetinic acid from Glycyrrhiza glabra and their derivatives could inhibit 3CL pro . 10 During our search for novel SARS-CoV 3CL pro inhibitors from medicinal plants, we found that the MeOH (95%) extracts from the bark of Tripteryguim regelii (Celastraceae) can remarkably inhibit SARS-CoV 3CL pro activity (>70% inhibition at 30 lg/mL). Subsequent bioactivity-guided fractionation of the CHCl 3 extracts led to the isolation of four quinone-methide triterpenoid derivatives 11 that were identified as celastrol (1), pristimererin (2), tingenone (3), and iguesterin (4) on the basis of their spectroscopic analyses (Fig. 1 ). 12 T. regelii is a woody vine native to Eastern and Southern China, Korea, Japan, and Taiwan. 13 This plant known as 'Thunder God Vine' has been historically used in traditional Chinese medicine to treat inflammatory and autoimmune diseases, such as rheumatoid arthritis (RA), systemic lupus erythematosus, psoriatic arthritis, and Behcet's disease. 14 A number of triterpenes, diterpenes, and sesquiterpenes as a major compound have been isolated from the bark of T. regelii and are known to have a variety of biological activities with good cytotoxicity to numerous cancer cell lines and efficacy in several rodent models of arthritis and 0960-894X/$ -see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.bmcl.2010.01.152

Abbreviations: IC 50 , the inhibitor concentration leading to 50% activity loss; K i , inhibition constant; K m , Michaelis-Menten constant; V max , maximum velocity; NMR, nuclear magnetic resonance. other inflammatory disease. 15 However, their SARS-CoV 3CL pro inhibitory activities have not been reported. In this study, we wish to describe the SARS-CoV 3CL pro inhibitory activities of the four quinone-methide triterpene derivatives 1-4 and semi-synthetic dihydrocelastrol 5.

The enzymatic activity of SARS-CoV 3CL pro was measured by a modification of the method described by Kuo et al. 16 The biological activities of 1-5 were assessed against SARS-CoV 3CL pro and confirmed by the positive control with curcumin 9 which inhibited SARS-CoV 3CL pro with IC 50 value of 23.5 lM. We found that the IC 50 values of the 1-5 ranged from 2.6 to 21.7 lM against SARS-CoV 3CL pro (Table 1) and that all were dose-dependent (Fig. 2) . Celasterol (1) having acid moiety at C-20 inhibited SARS-CoV In order to confirm whether quinone-methide moiety plays to inhibit SARS-CoV 3CL pro , celastrol (1) was reacted by dehydrogenation under Pd catalyst to give the dihydrocelastrol (5) in a quantitative yield, as shown in Scheme 1. According to Schwalbe's result, 17 celastrol (1) possesses electrophilic sites within the A and B rings, where nucleophilic groups of cysteine thiol group of cell division cycle protein 37 (Cdc37) reacts with the quinonemethide of 1 through a Michael addition, resulting in the formation of an adduct at C6. Thus, we believe that this moiety would be valuable for enhancing the inhibitory activity of 3CL pro . In fact, our result showed that quinone-methide moiety of 1 (IC 50 = 10.3 lM) increased the potency of SARS-CoV 3CL pro inhibition by twofold when compared with the reduced analogue 5 (IC 50 = 21.7 lM) ( Table 1 ). In view of this result, the presence of a quinone-methide moiety appears to play a relatively significant role in inhibition.

We then further characterized the inhibitory mechanism of the isolated quinone-methide triterpenes against SARS-CoV 3CL pro activity. This began with analysis of the mode of inhibition using both Lineweaver-Burk and Dixon plots. As shown representatively in Figure 3 , the kinetic plots show that compound 4 has a competitive inhibition mode of action. Because the Lineweaver-Burk plot of 1/V versus 1/[S] result in a family of straight lines with the same y-axis intercept, V max (44.4 ± 6.2 intensity min À1 ), respectively, for the SARS-CoV 3CL pro inhibitor 4 (Fig. 3A) . All isolated inhibitors manifested the same inhibition mode of action. The K i value of the most potent compound 4 was determined to be 0.8 lM (Table   1 ), as found from the common x-axis intercept of lines on the corresponding Dixon plot (Fig. 3B) . To better investigate the interaction of these inhibitors with the enzyme, we attempted a molecular docking simulation of the binding of 4 and 5 into SARS-CoV 3CL pro active site. The X-ray structure of the SARS-CoV 3CL pro in complex with a substrate-analogue inhibitor (coded 1uk4 or 2z3e) 18, 19 obtained from the Protein Data Bank (PDB; http://www.rcsb.org/pdb/) was used for modeling analysis. Computer docking analysis 20 revealed that iguesterin (4) can fit well into the substrate-binding pocket of SARS-CoV 3CL pro . As shown in Figure 4A , the hydroxyl group of C3 of 4 formed a hydrogen bond with the oxygen atom of the carbonyl group of Cys 44 and OH of Thr 25 located in domain I (residues 8-101), 9 whereas dihydrocelastrol (5) did not form any intermolecular bonds with the enzyme besides the hydrophobic interaction ( Fig. 4B) . Iguesterin (4) binding mode has different from betulinic acid (His164 and Thr24) and savinin (Cys145, Gln166, Gln189, Gly143 and Ser144) with 1uk4. These differences of intermolecular interaction apparently are reflected by the 10-fold smaller IC 50 value of iguesterin (4) compared with that of reference compounds in inhibiting the 3CL pro . Moreover, the calculated binding energy (kcal/mol) 19 of all quinone-methide triterpenes are shown in Table  2 . These docking experiments support the observation in the enzymatic assay, which reveals the important roles of the quinone-methide moieties inhibition of the SARS-CoV 3CL pro . Although this preliminary molecular modeling study cannot be final proof for this critical argument, it constitutes a positive sign for the possibility of the mechanism. In summary, we have identified quinone-methide triterpene derivatives as novel SARS-CoV 3CL pro inhibitors. The newly identified bioactive compounds with SARS-CoV 3CL pro inhibitory activity in the lM range include celastrol (1), pristimerin (2), tingenone (3) and iguesterin (4) . Although all isolated quinone-methide triterpenes has previously been known compounds, this is the first time it has been shown to possess SARS-CoV 3CL pro inhibitory activity. We are hopeful that these preliminary data may be able to open new avenues for research targeted toward reducing the threat of SARS to the global community. Further research and drug development on quinone-methide triterpenes as promising SARS-CoV 3CL pro inhibitors are ongoing in our laboratory and the results will be reported in due course. 

The organic layer was washed with brine, dried over anhydrous Na 2 SO 4 , and concentrated to give a dark brown residue (310 g) which was chromatographed on a silica gel column. The resultant extract (310 g) was suspended in H 2 O (2 Â 1 L). The diluted with H 2 O has been partitioned with organic solvents (CHCl 3 and BuOH) of the different polarities to afford CHCl 3 (162 g), BuOH (125 g), and H 2 O (150 g) extracts, respectively. The CHCl 3 and BuOH extracts were subjected to column chromatography using silica gel with hexane-acetone gradient and hexane-EtOAc gradient. The CHCl 3 extract (162 g) was subjected to column chromatography (glass column 10 Â 80 cm) over silica gel (1.5 kg; 70-230 mesh; Merk), eluted with gradient mixtures of hexane (4 L) and hexaneacetone, of increasing polarity (30/1?1/6), and finally with MeOH. Fifteen pooled fractions (fr.1-fr.30) were obtained after combining fractions with similar TLC profiles from this initial column chromatography. The column was eluted with solvents of increasing polarity (CHCl 3 -acetone) to give 30 fractions. Fraction 5 (14.3 g) was chromatographed on a silica gel column with hexane-acetone to give 45 fractions (5.1-5.30). The fractions from 5.11-5

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318, 862; (b) Briefly, a fluorogenic peptide Dabcyl-KNSTLQSGLRKE-Edans is used as the substrate, and the enhanced fluorescence due to cleavage of this substrate catalyzed by the protease was monitored at 590/20 nm with excitation at 360/40 nm. The IC50 value of individual compound was measured in a reaction mixture containing 10 lg/mL of the 3CL pro (final concentration was 2.5 lg) and 10 lM of the fluorogenic substrate in 20 mM Bis-Tris buffer. Reactions were run at 25°C with continuous monitoring of fluorescence for 60 min. Kinetic parameters were obtained using various concentrations of FRET peptide in the fluorescent assay. The maximal velocity

Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bmcl.2010.01.152.