GLI1 Inhibitors Identified by Target Protein Oriented Natural Products

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GLI1 Inhibitors Identified by Target Protein Oriented Natural Products Isolation (TPO-NAPI) with Hedgehog Inhibition Midori A. Arai, Fumie Ochi, Yoshinori Makita, Tetsuhiro Chiba, Kyohei Higashi, Akiko Suganami, Yutaka Tamura, Toshihiko Toida, Atsushi Iwama, Samir K. Sadhu, Firoj Ahmed, and Masami Ishibashi ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.8b00492 • Publication Date (Web): 30 Aug 2018 Downloaded from http://pubs.acs.org on August 31, 2018

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ACS Chemical Biology

GLI1 Inhibitors Identified by Target Protein Oriented Natural Products Isolation (TPO-NAPI) with Hedgehog Inhibition

Midori A. Arai,*† Fumie Ochi,† Yoshinori Makita,† Tetsuhiro Chiba,‡ Kyohei Higashi,∥ Akiko Suganami,‡ Yutaka Tamura,‡ Toshihiko Toida,† Atsushi Iwama,‡,⊥ Samir K. Sadhu,# Firoj Ahmed∇ and Masami Ishibashi*†

†

Graduate School of Pharmaceutical Sciences, Chiba University, 1-8-1 Inohana, Chuo-

ku, Chiba 260-8675, Japan. ‡

Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-

8670, Japan. ∥ Faculty

of Pharmaceutical Sciences, Tokyo University of Science, 2641 Yamazaki, Noda,

Chiba 278-8510, Japan. ⊥ The

Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku,

Tokyo 108-8639, Japan. # ∇

Pharmacy Discipline, Khulna University, Khulna-9208, Bangladesh. Department of Pharmaceutical Chemistry, University of Dhaka, Dhaka-1000,

Bangladesh.

E-mail: [email protected], [email protected]

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ACS Chemical Biology 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ABSTRACT This report describes a development of target protein oriented natural products isolation (TPO-NAPI) method for Hedgehog inhibitors and the direct GLI1 inhibitor, 5’-O-methyl3-hydroxyflemingin A (3), which inhibited hedgehog (Hh) signal transduction and diminished characteristics of cancer stem cells. Eight natural products (including three newly described products) that directly bind to GLI1 were rapidly obtained via TPONAPI method developed using GLI1 protein-immobilized beads. 5’-O-Methyl-3hydroxyflemingin A (3) inhibited Hh signaling (IC50 7.3 µM), leading to decreasing production of the Hh target proteins BCL2, PTCH1, and BMI1. 5’-O-Methyl-3hydroxyflemingin A (3) was cytotoxic to Hh-related cancer cells. CD experiments revealed that 5’-O-methyl-3-hydroxyflemingin A (3) directly bound GLI1 (Kd 7.7 µM). Moreover, 5’-O-methyl-3-hydroxyflemingin A (3) diminished cancer stem cells characters of Huh7 such as sphere formation and production of the cancer stem cell marker EpCAM.

These results suggest that Hh inhibitors can efficiently suppress the

activity of cancer stem cells.


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Hedgehog (Hh) signaling plays essential roles in development, such as stem cell maintenance and fate as well as cell proliferation, differentiation, and homeostasis in numerous tissues and organs.1,2 However, mutations in primary components of the Hh signaling pathway are associated with many human developmental disorders and cancers. In the OFF state, the protein patched 1 (PTCH1) suppresses the localization of smoothened (SMO) to cell surface of primary cilium (Figure 1). Members of the GLI (glioma-associated oncogene) family of transcription factors are phosphorylated by protein kinase A (PKA), casein kinase 1a (CK1a), and glycogen synthase kinase 3b (GSK3b). Proteolytic cleavage of phosphorylated GLIs in the proteasome generates the inactive forms of GLIRs. GLI3R inhibits the transcription of Hh target genes. In the ON state, Hh ligand binds to its receptor, PTCH1, reversing the inhibition of SMO. Active SMO translocates to the primary cilium to activate GLIs. Active GLIs in turn translocate to the nucleus to activate the transcription of various target genes, such as GLI1, PTCH1, BCL2 (B-cell leukemia/lymphoma 2), BMI1 (B cell–specific Moloney murine leukemia virus insertion region 1), cyclin D, cyclin E, and N-Myc. Hh signaling is involved in the proliferation of several types of cancer.3 Because Hh signaling is important in stem cell maintenance, the role of Hh signaling in cancer stem-like cells (CSCs) is the focus of increased research attention.4,5 CSCs can be



identified by distinct cell surface markers, such as BMI1, CD133, and CD44.6 The key stem cell regulatory gene BMI1 was recently shown to be directly controlled by Hh signaling.7 GLI1 binds to the promoter region of BMI1 to enhance the gene’s transcription. Because CSCs are capable of self-renewal, thus maintaining tumor growth and potentially generating different tumor cell types, strategies to target CSCs have received considerable recent research attention. Hh signaling inhibitors would be good candidates for use in cancer treatment. Most reported Hh inhibitors are SMO antagonists. Cyclopamine (isolated from Veratrum californicum8,9) CUR61414,10 SANTs,11 and the first FDA-approved Hh inhibitor, vismodegib,12 act on SMO. In addition, the Hh acyltransferase inhibitor RU-SKI 43,13 Hh protein binding inhibitor Robotnikinin,14 GLI1 modification inducers GANTs15, and AAA+ ATPase motor cytoplasmic dynein inhibitors HPIs16 have been reported. Using an originally developed cell-based assay system, our group has also identified many Hh inhibitors (Figure 2A), including physalin B,17 physalin F,17 colubrinic acid,18 caldenolides,19 taepeenin D,20 flavonoid glycoside,21 vitetrifolin D,22 physalin H,23 withaferin A,24 aciculatin,25 and synthetic chromones26. We were the first to demonstrate that vitetrifolin D, physalin H, and withaferin A directly inhibit the GLI1-DNA complex. Another GLI1-DNA complex inhibitor, glabrescione B, has also been reported.27


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Because GLI1 functions in the final step of Hh signaling, small molecules that directly inhibit GLI1 are attractive. To identify such direct-target protein inhibitors, we recently developed the target protein oriented natural product isolation (TPO-NAPI) method to screen natural products.28-30 In order to quickly identify GLI1 inhibitors, we newly developed the TPO-NAPI method using GLI1-immobilized beads (Figure 2B). Although a reporter assay screening is very efficient to obtain hit compounds, sometimes there are false positives which suppress gene expression non-specifically. If a new second screening was combined, the number of false positive would be reduced. To that aim, we designed the TPO-NAPI with GLI1 beads to obtain GLI1 inhibitors. Here, we report an efficient system for isolating GLI1 inhibitors using a combination of a reporter assay and the TPO-NAPI method with GLI1 beads.

RESULTS AND DISCUSSION TPO-NAPI of Naturally Occurring GLI1 Inhibitors. The first screening used a cell-based reporter assay system (Figure 2A) originally developed by our group.17 In the absence of tetracycline (Tc), expression of GLI1 was suppressed by the Tc repressor (TetR). Binding of Tc to the TetR releases inhibition, leading to exogenous GLI1 expression. GLI1 translocates to the nucleus and binds GLI



binding sites to induce expression of luciferase. If test compounds are Hh inhibitors, luminescence would decrease. Using this method, we first screened our Thailand (603) and Bangladesh (447) plant extract library to identify extracts including Hh signal inhibitors. After removal extracts which don’t have peaks of UV absorption, 655 plant extracts were screened by a reporter assay. The first screening hit extracts (92) were then screened using the TPO-NAPI system developed as described below. By using the combination screening, the number of extracts for second screening was reduced efficiently. In addition, this combination screening reduced the false positives. For example, cycloheximide showed potent inhibitory activity in GLI1 reporter assay (data not shown). However, this compound was known as a protein synthesis inhibitor. Cycloheximide was removed from hit compounds after second screening of GLI1-beads. A schematic illustration of the approach for identifying GLI1-binding natural products is shown in Figure 2B. We decided to use GLI1 partial protein that binds to DNA sequences containing five zinc finger domains (Supplementary Figure 1). Glutathione-Stransferase (GST)-fused GLI1-immobilized (GST-GLI1) beads were prepared by mixing GST-GLI1 and glutathione sepharose 4 beads (GE Healthcare). Because glutathione is a substrate of GST, we obtained tight binding between GST and glutathione. The concept is illustrated in Figure 2B. GLI1 beads were mixed with our natural product extract library


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and incubated at 4°C for 2 h. After incubation, the beads were washed to remove unbound natural products. Compounds bound to GLI1 are released by incubation in 70% EtOH at rt for 10 min and then analyzed by HPLC. Data regarding retention time, UV absorption pattern, and MS spectra were acquired, enabling us to separate the desired natural product components during fractionation and isolation steps. To eliminate compounds that bind non-specifically, GST-immobilized beads were used as in control experiments. Peaks present in GST-GLI1 bead results but not GST results represent GLI1-binding compounds. For these assays, comparable concentrations were used: GST-GLI1 beads (GST-GLI1; c.a. 3.6 nmol/bed volume 100 µL of beads, GST; c.a. 3.8 nmol/bed volume 100 µL of beads). To optimize the experimental conditions, first we screened our natural product library and found that arcyriaflavin B (1)31 bound the GLI1 beads and inhibited Hh signaling (IC50 0.61 µM). Incubation time, buffer, detergent, and method for releasing the bound compound from the GLI1 beads were optimized to develop the final GLI1-beadsHPLC method (Supplementary Figure 5). Using the reporter and GLI1-beads assays, 92 tropical plant extracts were screened. The results for Flemingia congesta (leaves) and Piper chaba (underground parts) collected in Bangladesh are shown in Figure 3A. Five hit peaks exhibiting obvious differences between GLI1 beads and GST beads were detected for F. congesta, and two



hit peaks were detected for P. chaba. Hit peaks A-F were isolated using column chromatography. The isolated compounds were identified as 3-hydroxyflemingin A (2) (peak A; novel compound), 5’O-methyl-3-hydroxyflemingin A (3) (peak B)32, 5’-O-methyl-3,4,2’,4’-tetrahydroxy-3’geranyldihydrochalcone (4) (peak C; novel compound), 5’-O-methylflemingin C (5) (peak D)32, 3,5’-dihyroxyflemingin A (6) (peak E; novel compound), piperine (7)33 (peak G), and pellitorine (8)33 (peak F). The structures of 2, 4, and 6 were determined by 1Hand 13C-NMR, HRMS, HMBC, COSY, and NOE (Figure 3B and Supporting Information). Compound 2 was isolated as yellow oil. The HR-ESI-MS showed a pseudo molecular ion peak [M+Na]+ at m/z 429.1637 (calcd for C25H26O5Na, ∆ -4.1 mmu), which indicated the molecular formula, C25H26O5. The IR absorption at 3338 and 1652 cm-1 indicated the presence of OH and C=O group, respectively. From the comparison of the 1H-NMR spectrum of known compound 3, the absence of MeO group at 5’ position was deduced. From other NMR data, the structure of 2 was determined as shown in Figure 3C. Compound 6 was isolated as yellow solid, and the HR-ESI-MS showed a pseudo molecular ion peak [M+H]+ at m/z 423.1805 (calcd for C25H27O6, ∆ -0.3 mmu). The similarity of the 1H-NMR spectrum with compound 3, the replacement of MeO group with OH group was deduced. The structure was determined based on other NMR data.


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Compound 4 was isolated as orange oil, and the HR-ESI-MS showed a pseudo molecular ion peak [M+Na]+ at m/z 461.1905 (calcd for C26H30O6Na, ∆ -3.5 mmu). Compared with 1

H-NMR spectrum of known compound 3, a vinyl proton at 1” position was disappeared

and methylene group appeared at the high field. In

13

C-NMR, the quaternary carbon

signal of 3 at 3” position was not detected. From the other NMR data, the structure of 4 was determined as shown Figure 3C. Enantiomeric isomers of compound 3 were isolated using chiral HPLC (CHIRALPACK AD-RH). By comparing CD spectra of reported similar compounds, the 3’’ position was determined as R for (–)-3 and S for (+)-3 (Figure 3C and Supplementary Figure 4). The ratio of (–)-3 and (+)-3 was 35 : 65. Interestingly, compounds 2, 5 and 6 were also mixture of their enantiomers (2; 79 : 21, 5; 40 : 60, 6; 46 : 54) (CHIRALPAK AD-RH, 90% MeOH+0.1% TFA, 1.0 ml min-1). The ability of the isolated compounds (2-8) to bind GLI1 was verified using GST-GLI1 beads (Supplementary Figure 5). With these isolated GLI1-binding natural compounds in hand, their ability to inhibit Hh signaling was examined using a cell-based reporter assay (Figure 4). Reporter cells in which the luciferase construct with GLI binding site was stably incorporated were treated with each compound for 12 h. After incubation, luciferase activity was measured. Simultaneously, the viability of sample-treated cells was measured using a fluorometric



microculture cytotoxicity assay (FMCA).34 The IC50 values of 2-8 were 9.1, 7.3, 14.2, 7.2, 9.2, 135.8, and 188.4 µM respectively. The enantiomers of 3 showed comparable activity: (–)-3, 5.9 µM; (+)-3, 5.8 µM. Therefore, the stereochemistry of the 3’’ position does not affect Hh inhibitory activity. Compound 4 appears to be a precursor of compound 3. Comparison of the activity of 4 and 3 suggested the pyran ring is important for activity. Since compound 3 and 5 have better activity than that of 2, addition of OMe group seems to give a good effect. Because there is no difference between 3 and 5 in the inhibitory activity, the position of the OH group on the right ring does not affect the activity.

5’-O-Methyl-3-hydroxyflemingin A (3) inhibits formation of the GLI1-DNA complex. Next, we examined the bioactivity of 5’-O-methyl-3-hydroxyflemingin A (3).35 First, we investigated the binding of 3 to GDT-GLI1 (171-515 aa) using CD measurements (Figures 5A, 5B). Many reports describe determination of Kd values using CD spectra after binding of ligand to protein.36-38 The CD spectra of GST-GLI1 (171-515 aa) and GST were analyzed from 205 to 250 nm in the presence of different concentrations of 3; shifts in magnitude at wavelength 208 nm reflecting an a-helical structure were then plotted. The CD values changed in the presence of 3, indicating that 3 induces a conformational change in GST-GLI1 upon binding. There was no significant change in


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GST. The Kd value for 3 and GST-GLI1 (171–515 aa) determined from double-reciprocal plots was 7.7 µM. Inhibition of the GLI1-DNA complex was investigated using an electrophoretic mobility shift assay (Figure 5C). The same GLI1 with five zinc finger domains used in TPO-NAPI experiments was used here. The DNA sequence contained the GLI1 binding site and a biotin tag. The GLI1-DNA complex appeared on the upper portion of the gel. As shown in Figure 8, compound 3 disrupted formation of the GLI1 and DNA complex.

Docking study of the binding of 3 to GLI1. To confirm Hh inhibitory activity and the results of CD measurements, we investigated the molecular basis of the interaction of compound 3 (R for [–]-3 and S for [+]-3) in the GLI1/DNA complex. Prior to the docking simulation, we set the interaction site of compound 3 on GLI1 and the residues (Lys350 and Arg354) at the N-terminus of the zinc finger 5 (ZF5) helix (a-helix) according to the report of Infante et al.27 As shown in Figure 6, both R- and S-3 interacted with the side chain of Lys350, a key residue in determining GLI1/DNA binding and GLI1 transcriptional activity, with almost the same interaction energy (R: –80.2 kcal/mol, S: –77.9 kcal/mol). Therefore, the chirality of the 3’’ position of compound 3 does not appear to affect the interaction with



GLI1, consistent with the results of Hh inhibitory activity analyses (R: IC50=5.9 µM, S: IC50=5.8 µM). Moreover, the interactions of both R- and S-3 with the side chain of Lys350, located on the ZF5 helix of GLI1, were consistent with the a-helical conformational change observed in the CD spectroscopic measurements, as described above.

5’-O-Methyl-3-hydroxyflemingin A (3) is cytotoxic to Hh-related cancer cells and down-regulates Hh-related protein expression. As aberrantly activated Hh signaling contributes to cancer development, we examined the cytotoxicity of 3 against various Hh-related cancer cell lines: human prostate (DU145), human pancreatic (PANC-1), and human hepatocellular carcinoma (Huh7) cells (Figure 10). We also examined the cytotoxicity of 3 toward normal C3H10T1/2 cells, which do not rely on the Hh ligand for survival (Figure 7A). Compound 3 showed potent cytotoxicity against DU145, PANC1, and Huh7 cells, with IC50 values of 9.4, 9.0, and 6.9 µM, respectively. Although 3 was also cytotoxic to C3H10T1/2 cells, the cytotoxicity against cancer cells was slightly stronger. As compound 3 was most effective against Huh7 cells, Hh-related protein levels were assessed (Figure 7B). As expected, expression of the Hh-related proteins anti-apoptotic protein BCL2, Hh receptor PTCH, and polycomb gene product BMI1 decreased.


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5’-O-Methyl-3-hydroxyflemingin A (3) inhibits cancer stem cells character of Huh7. BMI1 plays a crucial role in many tissues and organs in the self-renewal of a variety of somatic stem cells, such as haematopoietic,39 neural,40 and intestinal stem cells.41 A critical relationship between BMI1 and cancer stem cells (CSCs) has also been reported.42 Increased BMI1 expression is associated with poor clinical outcome in cancer patients.43 A recent study revealed that reducing BMI1 levels effectively suppresses breast CSCs.44 Therefore, therapies for inhibiting BMI1 expression would be very attractive in the treatment of cancer.45 Although there are several reports of treating CSCs with Hh inhibitors,46 reports of the suppression of characteristics of CSCs by Hh inhibitors are limited. We therefore investigated the inhibition of Huh7 cell sphere formation by compound 3. As BMI1 plays a critical role in the self-renewal of Huh7 cells,42,47,48 Hh inhibitor 3 should suppress sphere formation by these cells. As shown Figure 8A, sphereforming activity decreased in a dose-dependent manner upon treatment with compound 3. This result indicated that compound 3 inhibits the anchorage-independent growth of CSC-like Huh7 cells. The surface molecule EpCAM is a known CSC biomarker. A small population of Huh7 cells that highly express EpCAM (EpCAM-positive cells) exhibited features of CSCs.49 Compound 3 significantly decreased the EpCAM-positive fraction of



Huh7 cells after 24 h of treatment, from 91.4 to 85.6 and 51.6% at 15 and 25 µM, respectively (Figure 8B). These observations suggest that compound 3 suppresses CSC characteristics of Huh7 cells by inhibiting their growth and BMI1 expression.

Conclusions In conclusion, a new TPO-NAPI method for identifying Hh inhibitors was developed using GLI1-immobilized beads and HPLC. Three newly isolated natural products and five known compounds were identified as GLI1-binding compounds. This method is useful for identifying GLI1 inhibitors. 5’-O-Methyl-3-hydroxyflemingin A (3) (Hh reporter assay inhibition, IC50 7.3 µM) inhibited GLI1-DNA complex formation. Moreover, compound 3 suppressed various characteristics of Huh7 CSCs via inhibition of BMI1 expression. We thus believe that strategies using TPO-NAPI to identify GLI1 inhibitors will yield good candidates for development as anti-cancer drugs.

METHODS A typical screening procedure GST-GLI1 (171–515 aa) (234 µg, ca. 3.6 nmol) in PBS was mixed with glutathione sepharose 4B beads (bed volume 100 µL, GE Healthcare) at 4 °C for 1 h on a rotating


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mixer. The GST-GLI1 (171–515 aa) beads were washed four times with NT buffer (20 mM Tris-HCl [pH 7.5], 200 mM NaCl) and suspended in NT buffer (250 µL). To the freshly prepared GST-GLI1 (171–515 aa) beads (bed volume 100 µL), an extract of natural resources (187.5 µg in 50% EtOH, 37.5 µL) or compound (75 nmol in DMSO, 37.5 µL) was added and gently mixed for 2 h at 4 °C on a rotating mixer. The beads were then washed two times on a rotating mixer at 4 °C for 10 min with NT-N buffer (NT buffer containing 0.05% Nonidet P-40, 500 µL). Next, 70% EtOH (150 µL) was added to the beads, and the suspension was mixing at room temperature for 10 min. After centrifugation (2000 rpm, 4 °C, 1 min), the supernatant was centrifuged at 15000 rpm for 15 min. One-third of the supernatant was analyzed by HPLC. Control GST beads were prepared using the same procedure used to prepare GST-GLI1 (171–515 aa) beads. GST (100 µg, ca. 3.8 nmol) in PBS was added to pre-washed glutathione sepharose 4B beads (bed volume 100 µL, GE Healthcare). Extracts exhibiting an obvious difference in peak intensity between the GST-GLI1 (171–515 aa)-beads and GST-beads (control) were designated “hit” extracts containing GLI1-binding natural products.

Extraction of Flemingia congesta Air-dried leaves of F. congesta (160 g) were extracted with MeOH overnight at rt, and



the resulting extract (2.5 g) was subjected to chromatography on a Diaion HP-20 column (f 80 × 180 mm) eluted with a gradient of MeOH-acetone (1/0–0/1) to give five fractions (frs. 1A–1E). Fractions 1B and 1C (504.0 mg) were subjected to chromatography on an ODS column (40 × 200 mm) eluted with a gradient of H2O-MeOH (3/7–0/1) to give seven fractions (frs. 2A–2G). Fraction 2D (190.6 mg) was chromatographed on a DIOL silica gel column (f 30 × 200 mm) eluted with hexane/EtOAc (3/1–0/1) mixtures of increasing polarity and MeOH to yield compounds 2 (7.0 mg), 3 (43.7 mg), and 4 (15.7 mg). Fractions 1A, 2B, and 2C (871.2 mg) were subjected to chromatography on an ODS column (f 40 × 170 mm) eluted with a gradient of H2O-MeOH (3/7–0/1) to give nine fractions (frs. 4A–4I). Fraction 4D (44.8 mg) was chromatographed on a DIOL silica gel column (f 15 × 250 mm) eluted with hexane/EtOAc (7/4–1/1) mixtures of increasing polarity and MeOH to give five fractions (frs. 6A–6E). Fraction 6B (15.6 mg) was purified on a COSMOSIL 5C18-AR-II column (f 10 × 250 mm; eluent H2O/MeOH [1/4]; flow rate 2.0 mL min-1; UV detection at 350 nm) to yield 5 (1.2 mg, tR 34.0 min). Fraction 12C (6.2 mg) was purified on a COSMOSIL Cholester column (f 10 × 250 mm; eluent H2O/MeOH [1/9], flow rate 2.0 mL min-1; UV detection at 254 nm) to yield 6 (3.3 mg, tR 23.8 min). Compound 3 was a mixture of enantiomers in an approximate ratio of 65:35.


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Chiral preparative HPLC of 3 was conducted using a CHIRALPACK AD-RH column (f 1.0 × 150 mm; eluent H2O/MeOH [1/9], flow rate 1.0 mL min-1; UV detection at 350 nm).

Extraction of Piper chaba Air-dried underground parts of P. chaba (71.7 g) were extracted with MeOH overnight at rt, and the resulting extract (5.6 g) was suspended in H2O-MeOH (100/10, 110 mL) and partitioned with hexane (110 mL × 3), EtOAc (110 mL × 3), and nBuOH (110 mL × 3). The hexane-soluble fraction (1.19 g) was subjected to chromatography on a silica gel column (f 40 × 300 mm) eluted with gradient mixtures of hexane-EtOAc (9/1–0/1) and MeOH to give 17 fractions (frs. 1A–1Q). Fraction 1M (290.1 mg) was chromatographed on an ODS column (f 40 × 250 mm) eluted with H2O/MeOH (7/13) mixtures to yield 7 (132.2 mg). Fraction 1F (88.2 mg) was chromatographed on an ODS column (f 25 × 240 mm) eluted with H2O/MeOH (1/2–0/1) mixtures of decreasing polarity to yield 8 (40.4 mg). ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: XXXXXXXXXXXX.



Additional details of methods, isolation charts, NMR tables of compounds, NMR data of compounds (PDF)

ACKNOWLEDGEMENTS We are very grateful to F. Aberger and G. Regl (University of Salzburg) for providing tetracycline-regulated HaCaT cells; R. Toftgård (Karolinska Institute) for the 12GLI-RETKO luciferase plasmid; and B. Vogelstein (Howard Hughes Medical Institute) and H. Sasaki (RIKEN) for the human GLI1 plasmid. This study was supported by KAKENHI grants (numbers 18H02582, 17H03992, and 15H04650) from the Japan Society for the Promotion of Science, as well as the Takeda Science Foundation, Astellas Foundation for Research on Metabolic Disorders, the Naito Foundation, Strategic Priority Research Promotion Program, Chiba University, “Phytochemical Plant Molecular Sciences”, JSPS A3 Foresight Program, and the Workshop on Chirality at Chiba University (WCCU). This work was inspired by the international and interdisciplinary environment of the JSPS Core-to-Core Program “Asian Chemical Biology Initiative”.

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ON state (presence of Hh protein)

Page 28 of 44

primary cilium kinases*

SMO

SuFu GLI1/2/3

Hh GLI1/2/3A PTCH1

Transcription of target genes

nucleus SMO

GLI1/2/3A

GLI1, PTCH1 BCL2, BMI1

Figure 1 The hedgehog (Hh) signaling pathway. In the absence of Hh protein (OFF state), PTCH1 prevents SMO localization to the surface of primary cilium. GLIs are phosphorylated by kinases and then proteolytically cleaved. GLI3R supresses the transcription of Hh target genes. In the presence of Hh protein (ON state), the inhibition of SMO is released, and SMO localized to the surface of primary cilium to activate GLIs. GLI1/2/3A translocate to nucleus to induce the transcription of target genes. PTCH1: patched 1, SMO: smoothened, SuFu: suppressor of fused, PKA: protein kinase A, CK1a: casein kinase 1a, GSK3b: glycogen synthase kinase 3b, GLIR: inactive form of GLI, GLIA: active form of GLI.


Page 29 of 44

A) First screening Without

Tc

With

CMV prom.

Tc

CMV prom.

TetR

TetR

TetR

CMV prom.

TetR TetR 2

TetR

TetO2

TetR TetR Tc Tc

CMV prom.

GLI1

2

TetO2

GLI

Stop

GLI1

Luciferase

GLI GLI TK prom.

Luc.

12xGLI binding sites

B) Second screening

Hit compound extract wash

GLI1 GSH

GLI1

dissociation

HPLC

GST

GST

UV absorbance

GST-GLI1 beads

Retention time

Glutathione Sepharose 4B GST beads (control) extract dissociation

wash GST

GST GSH : glutathione (reduced form)

HPLC

UV absorbance

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Retention time

GST : glutathione S-transferase

Figure 2 Schematic illustration of screening method. A) Cell-based reported assay; without Tc, GLI1 expression is inhibited by TetR. With Tc, Tc binds to TetR to release the inhibition, leading to expression of excess amounts of GLI1. GLI1 binds to the GLI binding site to stimulate luciferase expression. Tc: tetracycline, CMV: cytomegalovirus promoter, TetR: tetracycline repressor, TetO: tetracycline operator, Luc: luciferase. B) Target protein–oriented natural product isolation using GLI1 beads. After incubation of the extract with GLI1 beads, washing and release steps gave hit compounds by HPLC. Experiments using GST beads were also performed as a control.



C

A Flemingia congesta

F

H N

O

Piper chaba

B

C

Page 30 of 44

OH

G

N H

N H

1

32

min

34

OH O 1’’ 2’ 3’ 1’ 3’’ O 4’ 5’ OMe

GST-GLI1 beads GST beads

GST-GLI1 beads GST beads 29

36

30 min

OH

O

2

E

30

O

OH

HO

A

D

O

31

1

OH

OH

3

OH

HO OMe

O

OH

OH

4 O

OH OH

O

O OMe

2

OH OH

3

B

O

OH

OH

OH

5

O

6

O O

N

N H

O

8

7 4 OH

O

OH

OH

O

OH

3’’ R

6

OH OH

3’’ O

OMe

S

(-)-3

O

OMe

(+)-3

Figure 3 (A) Results of screening using TPO-NAPI with GLI1-immobilized beads. Peaks exhibiting obvious differences between GLI1 beads and GST beads were subjected to isolation steps. (B) Key 1H-1H-COSY and HMBC data. (C) Structures of arcyriaflavin B (1) and other isolated natural products (2-7).


Page 31 of 44 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


2

(%)

3

(%)

100

100

50

50

50

0

0 5

7.5

5

10 12.5 (μM)

5

10 12.5 (μM)

5

10 15 25 (μM)

(-)-3

(%)

100

100

100

50

50

50

50

5

5

7.5 10 (μM)

(%) 150

7

7.5 10

5

15 (μM)

IC50 = 9.2 μM

IC50 = 7.2 μM (%)

0

0

0 2.5

(+)-3

(%)

100

0

viability

IC50 = 14.2 μM

6

(%)

luciferase

0

IC50 = 7.3 μM

IC50 = 9.1 μM (%)

7.5

4

(%)

100

7.5

10

IC50 = 5.9 μM

(μM)

5

7.5

10

(μM)

IC50 = 5.8 μM

8

150

100

100

50

50

0

100 125 150 (μM)

0

IC50 = 135.8 μM

100 150 200 (μM)

IC50 = 188.4 μM

Figure 4 Hh inhibitory activity of isolated compounds. GLI1 reporter cells were treated with each compounds for 12 h, after which luciferase activity was measured. Cell viability was assessed using FMCA.



A C 3 (μM) GLI1 DNA

+

+ +

10 + +

50 + +

100 + +

GLI1

DNA

B

200

Shift in ellipticity (degcm 2/dmol)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 32 of 44

Kd = 7.7 µM

150 100

free DNA

50 0 -50

0

20

40

60

3 (µM)

Figure 5 (A) Effects of 3 on CD spectra of proteins. A) CD spectra of GST-GLI1 (175515aa) and GST. (B) Concentration-dependent effects of 3 on the magnitude of the GSTGLI1 (175-515aa) response at 208 nm. CD measurements were performed as described in the Supporting Information. Values are mean ± SD. The Kd value of 3 was determined from double-reciprocal plots. (C) Electrophoretic mobility shift assay. GST-GLI1 protein (171–515 aa) and biotin-tagged DNA with the GLI1 binding site were incubated with or without compound 3. DNA sequence containing the GLI1 binding site (underlined): 5’AGCTACCTGGGTGGTCTCTTCGA-3’.


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R: (-)-3

S: (+)-3

Asp375

Asp375

Arg354

Arg354 Lys350

Figure 6 Docking analysis of the binding of 3 to GLI1.


Lys350


Page 34 of 44

A Cell line

IC50 (μM)

DU145

9.4

PANC1

9.0

Huh7

6.9

C3H10T1/2

10.4

log [comp. 3], M

B Huh7 cells 3 (μM)

0

1

5

10

1.00

0.84

0.81

0.71

BCL2 PTCH BMI1 β-actin

Figure 7 Effect of 3 on Hh-related cancer cell lines. A) Cytotoxic effects of 3 on DU145, PANC-1, Huh7, and C3H10T1/2 cells. Assays were performed in 0.05% DMSO (n = 3). Error bars represent SD. B) Inhibition of protein expression by compound 3. Western blot analysis of BCL2, PTCH, and BMI1 protein levels in Huh7 cells.


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A

control

3 (4 μM)

Number of spheres

3 (10 μM) 300 250 200 150 100 50

scale bar 100 μm

0 cont

1

4

10

(μM)

3

B

EpCAM(-)

EpCAM(+)

control 100%

EpCAM (-)

EpCAM (+)

8.6% 14.4% 48.4%

80% 60%

3 (15 µM)

40%

91.4% 85.6% 51.6%

20% 0% control

15 μM

25 μM

3

3 (25 µM)

Figure 8 (A) Assay of the effect of 3 on sphere-forming ability of Huh7 cells. Huh7 cells were incubated in low-adherence plates with or without of 3 for 7 days, after which the number of spheres with diameter >50 µm was determined. (B) Flow cytometry profiles of Huh7 cells incubated with or without compound 3 for 24 h.



Graphical Abstruct 180x180mm (300 x 300 DPI)

ACS Paragon Plus Environment

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105x105mm (300 x 300 DPI)



105x105mm (300 x 300 DPI)


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105x105mm (300 x 300 DPI)



105x105mm (300 x 300 DPI)


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105x105mm (300 x 300 DPI)



105x105mm (300 x 300 DPI)


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105x105mm (300 x 300 DPI)



105x105mm (300 x 300 DPI)


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GLI1 Inhibitors Identified by Target Protein Oriented Natural Products

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