Bioorganic & Medicinal Chemistry Letters 24 (2014) 2486–2492

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Biological evaluation of tanshindiols as EZH2 histone methyltransferase inhibitors Jimin Woo a,b, Hyun-Young Kim a, Byung Jin Byun a, Chong-Hak Chae a, Ji Young Lee a,c, Shi Yong Ryu a,c, Woo-Kyu Park a, Heeyeong Cho a,b,⇑, Gildon Choi a,b,⇑ a b c

Pharmacology Research Group, Drug Discovery Division, Korea Research Institute of Chemical Technology, Gajeong-Ro 141, Yuseong-gu, Daejeon 305-600, Republic of Korea Medicinal and Pharmaceutical Chemistry, University of Science and Technology, Gajeong-Ro 217, Yuseong-gu, Daejeon 305-350, Republic of Korea Graduate School of New Drug Discovery and Development, Chungnam National University, Daehak-ro 79, Yuseong-gu, Daejeon 305-764, Republic of Korea

a r t i c l e

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Article history: Received 19 November 2013 Revised 20 March 2014 Accepted 4 April 2014 Available online 13 April 2014 Keywords: Tanshinone Tanshindiol EZH2 Histone methyltransferase

a b s t r a c t EZH2 is the core subunit of Polycomb repressive complex 2 catalyzing the methylation of histone H3 lysine-27 and closely involved in tumorigenesis. To discover small molecule inhibitors for EZH2 methyltransferase activity, we performed an inhibitor screen with catalytically active EZH2 protein complex and identified tanshindiols as EZH2 inhibitors. Tanshindiol B and C potently inhibited the methyltransferase activity in in vitro enzymatic assay with IC50 values of 0.52 lM and 0.55 lM, respectively. Tanshindiol C exhibited growth inhibition of several cancer cells including Pfeiffer cell line, a diffuse large B cell lymphoma harboring EZH2 A677G activating mutation. Tanshindiol treatment in Pfeiffer cells significantly decreased the tri-methylated form of histone H3 lysine-27, a substrate of EZH2, as revealed by Western blot analysis and histone methylation ELISA. Based on enzyme kinetics and docking studies, we propose that tanshindiol-mediated inhibition of EZH2 activity is competitive for the substrate S-adenosylmethionine. Taken together, our findings strongly suggest that tanshindiols possess a unique anti-cancer activity whose mechanism involves the inhibition of EZH2 activity and would provide chemically valuable information for designing a new class of potent EZH2 inhibitors. Ó 2014 Elsevier Ltd. All rights reserved.

Histones are the small basic proteins found in eukaryotic cell nucleus, forming chromatin structures with DNA as the chief protein components. Covalent histone modifications such as phosphorylation, ubiquitination, acetylation and methylation play an important role in regulating chromatin dynamics and function.1 Enhancer of zeste homologue 2 (EZH2) is the core protein of Polycomb repressive complex 2 (PRC2) catalyzing primarily tri-methylation of histone H3 Lys-27 (H3K27). Tri-methylation of the lysine-27 has been suggested to cause the repression of specific genes, including many tumor suppressor genes.2 High EZH2 expression has been shown to be correlated with poor prognosis, high grade and high stage in several cancer types. In addition, several kinds of heterozygous mutations were found in ca. 7% of

Abbreviations: EZH2, enhancer of zeste homologue 2; ELISA, enzyme-linked immunosorbent assay; IC50, half maximal inhibitory concentration; GI50, half maximal growth-inhibitory concentration; Ki, dissociation constant of the enzyme– inhibitor complex; EED, embryonic ectoderm development; SUZ12, suppressor of zeste 12 homologue. ⇑ Corresponding authors. Tel.: +82 42 860 7426 (H.Cho), +82 42 860 7428 (G.Choi). E-mail addresses: [email protected] (H. Cho), [email protected] (G. Choi). http://dx.doi.org/10.1016/j.bmcl.2014.04.010 0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

follicular lymphomas and ca. 22% of diffuse large B cell lymphomas (DLBCL).3 The DLBCL cell lines with EZH2 mutation at position of Tyr-641 or Ala-677 are known to be very sensitive to EZH2 inhibitors, indicating that the cell lines are dependent on EZH2 activity for cell growth.4 Thus, blocking its gene expression or enzyme activity is considered to be a promising therapeutic strategy to treat cancers. Recently, small molecule inhibitors of EZH2 have been reported to produce promising anti-tumor activity both in vitro and in vivo.4 The root of Salvia miltiorrhiza (Danshen) has been widely used as traditional Chinese medicine for many years and shown to exhibit significant pharmacological activities for a variety of human diseases including cancers.5 Tanshinones, the major active components, belong to a group of an abietane-type diterpenes containing a 1,2-quinone in the C ring (Fig. 1). Both tanshinone I and tanshinone IIA have been shown to possess antitumor activities against several human cell lines,6 but display different activity and selectivity due to their structural differences.7 For example, tanshinone I inhibited migration and invasion of human lung adenocarcinoma cell line CL1–5 through reducing IL-8 expression, while tanshinone IIA induced cell differentiation and apoptosis.8

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Figure 1. Structures of tanshindiols and the related tanshinones.

The structural differences among tanshinones could explain why the individual tanshinones have unique biological activities different from each other. While more than 50 tanshinones have been identified from phytochemical study so far, many of the minor components including tanshindiols still need to be characterized whether they exhibit unique biochemical activity. Here, we report the inhibition of EZH2 histone methyltransferase activity by tanshindiols and its anticancer effects against cancer cell lines for the first time. In addition, mode of inhibition for EZH2 was also proposed based on the results from enzyme kinetic and docking experiments in present study. In an effort to discover new inhibitors for EZH2, we developed in vitro histone methyltransferase assay based on homogeneous, time-resolved fluorescence resonance energy transfer (TR-FRET).9 The catalytically active EZH2 protein complex containing EZH2, EED and SUZ12 was used for EZH2 activity measurements. We performed an inhibitor screen for natural product compounds against EZH2 activity as part of our ongoing EZH2 inhibitor program. Among the tested compounds, several diterpenes (1–6) illustrated in Figure 1 exhibited significant inhibitory effect on EZH2 activity in a dose-dependent manner. Dose–response curves of 1–6 were depicted in Figure 2 and the concentrations of 1–6 required for the 50% inhibitory effect on EZH2 activity (IC50 values) were summarized in Table 1. Tanshindiols B (4) and C (5), two stereoisomer

Figure 2. Inhibitory effects of several diterpenoids on EZH2 histone methyltransferases. EZH2 activity was measured in the presence of histone H3 peptide (150 nM) and SAM (3 lM) as substrates. Chemical structures of the compounds tested in this study are shown in Figure 1. Tanshindiols B and C (compounds 4 and 5) were found to be the most potent ones among the compounds tested (IC50 = 0.52 lM for tanshindiol B and 0.55 lM for tanshindiol C, respectively). The other tanshinones were significantly less active compared with tanshindiols. The IC50 values are also summarized in Table 1.

Table 1 Inhibition of EZH2 methyltransferase activity by tanshinones Number

Compound name

IC50 (lM)

1 2 3 4 5 6

Tanshinone I Tanshinone IIA Tanshinone IIB Tanshindiol B Tanshindiol C Methyl-tanshinonate GSK-126

8.9 ± 1.1 28.1 ± 5.8 4.8 ± 0.7 0.52 ± 0.09 0.55 ± 0.11 9.6 ± 1.3 0.016 ± 0.009

IC50: half maximal inhibitory concentration.

of each other with 1,2 diol moiety in A ring were found to be the most potent ones with IC50 values of 0.52 lM and 0.55 lM, while other structurally related tanshinones (1, 2, 3, 6) showed relatively weak inhibitory activity with IC50 values ranging from 4.8 to 28.1 lM (Table 1). These results strongly suggested that the abietane skeleton of tanshinones seemed to be essential for the inhibitory effect on EZH2 activity and 1,2 diol moiety in A ring of tanshinones could play a critical role for tanshinone compounds to bind to the active site of EZH2. However, it is not confirmative and needs more detailed SAR studies with various synthesized tanshinone derivatives, especially with other substituents in A ring. A detailed kinetic study was performed to examine the mode of inhibition of tanshindiol C by using the PRC2 complex as an enzyme source.9 EZH2 utilizes two substrates for catalysis, histone H3 and the methyl group donor, S-adenosylmethionine (SAM). As shown in Figure 3A, tanshindiol-mediated inhibition of EZH2 activity was found to be competitive for SAM with a Ki of 194 ± 24 nM, as indicated by increased Km values and unchanged Vmax values when the inhibitor concentration was increased. Meanwhile, tanshindiol-mediated inhibition of EZH2 activity seems to be a mixed inhibition for the peptide substrate as revealed by the simultaneous changes in the Km and Vmax values (Fig. 3B). Based on the observation of tanshindiol C as a SAM-competitive inhibitor (Fig. 3C), Ki value of the compound for the mutant PRC2 complex including A667G EZH2 was also calculated from the slopes and intercepts of Lineweaver–Burk plots, and determined as 176 ± 120 nM. These data suggest that tanshindiol C inhibits both wild-type and A667G mutant EZH2 activity with similar potencies. We next tested growth-inhibitory activity of tanshindiol C against various tumor cell lines. Cell growth inhibition was evaluated with WST-1 viability assay.10 As shown in Figure 4A, tanshindiol C inhibited growth of the cell lines such as Pfeiffer, U-2932 and Daudi (lymphoma), PC3 (prostate cancer), T98G and U87MG

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Figure 4. Growth inhibition of tumor cell lines by tanshindiol C (A) and GSK-126 (B). Effects of the compounds on the viability of several tumor cell lines were examined by WST-1 assay. Pfeiffer cell line was found to be the most sensitive one to tanshindiol C with a GI50 of 1.5 lM among the cell lines tested. GSK-126 also exhibited potent growth-inhibitory activity for Pfeiffer cells with a GI50 of 0.18 lM. The GI50 values are summarized in Table 2.

harboring heterozygous EZH2 A677G mutation. The A677G mutation is known to be a gain-of-function mutation enhancing the catalytic efficiency of EZH2, resulting in increased H3K27 tri-methylation.4 In contrast, U-2932 and Daudi lymphoma cell lines expressing wild-type EZH2 alone were found to be less sensitive to tanshindiol C with GI50 value of 9.5 lM and 10.6 lM, respectively. When growth-inhibitory activity of GSK-126, a recently reported EZH2 inhibitor, was examined against the cancer

Figure 3. Kinetic analysis of inhibition mode of tanshindiol C. (A) The PRC2 complex containing EZH2, EED, SUZ12 and RbAp46/48 was used as an enzyme source for EZH2 activity measurements. Lineweaver–Burk plot (L–B plot) of the effect of tanshindiol C on the activity of wild-type EZH2 at a fixed concentration of histone H3 peptide substrate (150 nM) and increasing concentrations of SAM. (B) L– B plot of the effect of tanshindiol C on wild-type EZH2 at a fixed concentration of SAM (3 lM) and increasing concentrations of histone H3 peptide. (C) L–B plot of the effect of tanshindiol C on the activity of A677G EZH2. The concentrations of tanshindiol C were 2.5 (j), 1.25 ( ), 0.625 ( ) and 0 lM (h), respectively. The Ki values of the compound were 194 ± 24 nM and 176 ± 120 nM, respectively, for the wild type and A677G EZH2 activity.

Table 2 Growth inhibition of various tumor cell lines by tanshindiol C

(glioma), and A549 (lung cancer). Among the cell lines tested, Pfeiffer was the most sensitive one to tanshindiol C with GI50 of 1.5 lM (Table 2). Pfeiffer cell line is a diffuse large B cell lymphoma

GI50: half maximal growth-inhibitory concentration. DLBCL: diffuse large B cell lymphoma. BL: Burkitt’s lymphoma.

GI50 (lM)

Cell line

Pfeiffer (lymphoma, DLBCL) U-2932 (lymphoma, DLBCL) Daudi (lymphoma, BL) PC3 (prostate cancer) T98G (glioma) U87MG (glioma) A549 (lung adenocarcinoma)

Tanshindiol C

GSK-126

1.5 ± 0.1 9.5 ± 0.3 10.6 ± 2.7 4.0 ± 0.1 6.0 ± 0.8 5.7 ± 0.5 4.2 ± 1.1

0.18 ± 0.02 6.7 ± 2.5 11.2 ± 0.7 9.4 ± 0.9 12.6 ± 1.8 28.5 ± 3.5 18.7 ± 0.8

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Figure 5. Apoptotic effects of tanshindiol C on Pfeiffer cells. (A) DNA content histograms of the cells treated with various concentrations of tanshindiol C for 72 h. The sub-G1 population was found to be increased upon the treatment of compounds (11%, 13% and 21% for 1 lM, 2.5 lM and 5 lM of tanshindiol C, respectively) relative to the control (9%). M1: sub-G1 phase, M2: G1 phase, M3: S phase, M4: G2/M phase (B) Western blotting of lysates from Pfeiffer cells that had been treated for tanshindiol for 72 h. The levels of cleaved forms of caspase-3, caspase-7 and PARP were found to be increased by tanshindiol C (1 lM and 3 lM) or GSK-126 (2 lM) treatments, indicating the cell death seems to be driven by apoptosis.

cell lines for comparison (Fig. 4B), Pfeiffer cell line was also found to be the most sensitive one to the inhibitor treatment with a GI50 of 0.18 lM, which is ca. 8.3-fold lower than that of tanshindiol C (Table 2). Proliferation of the DLBCL cell lines harboring EZH2 mutations such as Y641N, Y641F or A677G is known to be much more

sensitive to GSK-126 treatment than wild-type cell lines.4 This is attributed to the critical dependency on EZH2 activity for proliferation of these mutant cell lines, resulting in robust cell killing by the inhibitor treatment. In contrast, proliferation of the wild type cancer cells is not critically dependent on EZH2-mediated trimethylation of H3K27 and not affected strongly by the inhibitor

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Figure 6. Cellular mechanistic activity of tanshindiol C. (A) Changes in the level of tri-methylated H3K27 by tanshindiol C was examined in Pfeiffer cells by histone methylation ELISA. Results are expressed as means SEMs (n = 3). The amount of H3K27me3 treated with DMSO was expressed as 100%. ⁄⁄⁄P

Biological evaluation of tanshindiols as EZH2 histone methyltransferase inhibitors.

EZH2 is the core subunit of Polycomb repressive complex 2 catalyzing the methylation of histone H3 lysine-27 and closely involved in tumorigenesis. To...
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