Note pubs.acs.org/jnp
Inubosins A, B, and C Are Acridine Alkaloids Isolated from a Culture of Streptomyces sp. IFM 11440 with Ngn2 Promoter Activity Midori A. Arai,* Kazune Koryudzu, and Masami Ishibashi* Graduate School of Pharmaceutical Sciences, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8675, Japan S Supporting Information *
ABSTRACT: Three new acridine alkaloids, inubosins A (1), B (2), and C (3), were isolated from an extract of a culture of Streptomyces sp. IFM 11440 using bioassay-guided fractionation. Neurogenin2 (Ngn2) is an activator-type basic helix−loop−helix transcription factor that promotes neural stem cell differentiation. Using cell-based Ngn2 promoter activity-guided screening, Streptomyces sp. IFM 11440 was found to induce Ngn2 promoter activity. The structures of 1−3 were established using spectroscopic methods, including 1Dand 2D-NMR measurements. Inubosin B (2) showed potent Ngn2 promoter activity. Moreover, inubosin B (2) increased mRNA expression of genes related to neural stem cell differentiation.
N
eural stem cells have been identified in the subventricular zone of the lateral ventricle1 and the subgranular zone of the hippocampal dentate gyrus2 in the adult mammalian brain. Several reports have revealed that, after an insult, the adult mammalian brain shows increased neural stem cell differentiation to regenerate lost neurons.3−5 These reports showed that new therapies that promote neurogenesis in situ can improve nervous system disorders by providing an increase in new neurons. Small molecules that promote differentiation have received attention as possibly being useful in neural regenerative medicine for clinical treatment to increase neurogenesis of endogenous and/or transplanted neural stem cells. Basic helix−loop−helix (bHLH) activator and repressor genes regulate the timing of cell differentiation and the fate of neural stem cells.6−9 Neurogenin2 (Ngn2), a pro-neural bHLH transcriptional activator, forms a heterodimer with E proteins (e.g., E47) to bind the E box (CANNTG) in the regulatory regions of target genes to promote differentiation into neurons and glial cells. Therefore, Ngn2 promoter activators may accelerate the differentiation of neural stem cells. However, limited studies regarding natural and synthetic compounds as differentiation activators have been published.10−20 Recently, we reported the first search for differentiation activators from natural sources using Ngn2 promoter activity (Figure 1).20 In the present study, we isolated three new acridine alkaloids from an extract of a culture of Streptomyces sp. IFM 11440 using bioassay-guided fractionation. The Ngn2 promoter activity and promotion of neural stem cell differentiation induced by the isolated compounds were evaluated. We constructed a cell-based reporter assay for Ngn2 promoter activity using C3H10T1/2 mesenchymal progenitor cells that were stably transfected with a luciferase reporter containing the mouse Ngn2 promoter region (1650 bp). Screening for Ngn2 promoter activity was performed with our actinomycetes extract library using this high-throughput cell© XXXX American Chemical Society and American Society of Pharmacognosy
Figure 1. Two-step screening method for neural stem cell differentiation accelerators from extracts of natural resources.
based assay. As a result, an extract of a culture of Streptomyces sp. IFM 11440, which was isolated from a soil sample collected from Inubosaki, Choshi City in Chiba Prefecture, Japan, was found to contain an activator of the Ngn2 promoter. We also examined the cytotoxicity of the samples using a fluorimetric microculture cytotoxicity assay21 to rule out false-negative results caused by cytotoxicity of the test samples. The Streptomyces sp. IFM 11440 extract did not show cytotoxicity in this assay. The culture broth (24 L) of Streptomyces sp. IFM 11440 was harvested and centrifuged to separate the mycelium and the supernatant. Activity-guided separation of the supernatant by HP20 and ODS column chromatography led to the Received: August 4, 2014
A
DOI: 10.1021/np5006218 J. Nat. Prod. XXXX, XXX, XXX−XXX
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isolation of three new acridine alkaloids (Figure 2). Inubosin A (1) had a formula of C14H11NO2 as established by HRESIMS.
Figure 2. Structure of inubosins.
The 13C NMR spectrum displayed 14 carbon resonances (Table 1) including six quaternary carbon atoms, seven Figure 3. Structure determination of inubosins. (A) HMBC (red arrows) and COSY (blue lines). (B) NOEs.
Table 1. 1H and 13C NMR Data of Compounds 1−3 (600 MHz for 1H and 150 MHz for 13C; δ in ppm, J in Hz; in DMSO-d6) 1 position
a
δC
2 δH (J in Hz)
8.03 d (1.1)
δC
1
124.0
2 3
140.2 130.0
4
146.3
4a 5
147.6 152.3
6
109.6
7.10 d (7.2)
130.0
7
126.6
125.9
8
118.1
7.44 dd (8.3, 7.2) 7.58 d (8.3)
8a 9 9a 10a 11 OHa
127.1 135.4 126.3 140.2 62.9
7.78 dd (9.5, 1.1) 8.16 d (9.5)
113.9 141.3 109.1
9.00 s
4.73 brs 5.47, 9.80 brs
3 δH (J in Hz)
7.50 d (0.9) 7.07 d (0.9)
152.5 140.4 128.8
128.4 126.7 135.4 126.7 146.4 63.1
and the correlations observed in the 1H−1H COSY. Inubosin C (3) had the molecular formula C14H11NO3. Analysis of 1H and 13 C NMR data revealed the presence of an acridine ring, an oxymethylene, and three hydroxy groups. The structure of 3 was elucidated as shown in Figure 3. Inubosin C (3) has two phenolic hydroxy groups on both C-4 and C-5 positions, while inubosin A (1) and inubosin B (2) had only one hydroxy group on C-5 and C-4, respectively. The Ngn2 promoter activation by the isolated compounds was evaluated (Figure 4). Inubosin B showed potent, dosedependent promoter activity. The natural product baicalin, which is the only reported compound that can accelerate neural stem cell differentiation by increasing Ngn2 mRNA expression via a mechanism involving Erk1/2 activation, was used as a positive control.19 The activity of baicalin (100 μM) was 1.5 times higher than that of the negative control (DMSO) in our assay. Inubosin B (20 μM) showed 2.8 times higher activity than the control (DMSO), showing 1.9-fold greater activity than baicalin. Interestingly, the compounds inubosin A (1) and C (3), which are structurally similar to inubosin B (2), did not show significant activity. This result indicated that Ngn2 promoter activity requires a hydroxy group at the 4 position; however, a hydroxy group at the 5 position seems to reduce the activity. C17.2 neural stem cells can differentiate into neural cells, including neurons, astrocytes, and oligodendrocytes,25 and the effect of inubosins on differentiation was evaluated. Despite low Ngn2 promoter activity in the Ngn2 promoter assay, inubosin A (1) induced typical morphology indicative of differentiated cell bodies, similar to inubosin B (2) (data not shown). To quantitatively determine the activity of inubosins on differentiation, the expression of differentiation-related genes was investigated in C17.2 cells (Figure 5). Inubosin B (2) increased the mRNA expression of pro-neural transcriptional factors, such as Ngn2 and Ngn1, as expected. Interestingly, inubosin A (1) also promoted Ngn2 and Ngn1 mRNA expression. As inubosin A (1) did not show Ngn2 promoter activity, its mechanism may involve other events. Promotion activity of Ngn2 on NeuroD2 has been reported,26 and an increase in the expression of a neurogenic differentiation factor such as NeuroD2 after treatment with inubosin A (1) or B (2) is possible. Indeed, NeuroD2 expression was upregulated by these two compounds. Inubosin A (1) and B (2) also increased the mRNA level of the pro-neural neurotrophic growth factor neurotrophin 3 (NT3).27 These data indicated that inubosins A (1) and B (2) promoted neural stem cell differentiation by increasing the mRNA levels of relevant genes. To the best of our knowledge, inubosin B (2) is the first isolated new natural product that has
δC
δH (J in Hz)
113.6
7.51 d (0.9)
142.0 108.2
7.05 d (0.9)
152.5
8.19 d (8.5) 7.82 dd (8.5, 6.7) 7.60 dd (8.3, 6.7) 8.14 d (8.3) 8.99 s
4.64 brs 5.39, 9.82 brs
137.9 152.9 108.8
7.09 d (7.2)
127.2
7.47 dd (8.1, 7.2)
117.7
7.57 d (8.1)
127.3 135.1 126.9 137.9 63.2
8.96 s
4.65 brs 5.39, 10.12, 10.15 brs
These signals disappeared after addition of D2O.
methine carbon atoms, and one oxygenated methylene carbon atom. The 1H NMR spectrum indicated the presence of seven aromatic protons and one oxymethylene proton. The presence of two moieties with contiguous hydrogens for H-6/H-7/H-8 (δ 7.10, 7.44, and 7.58) and H-3/H-4 (δ 7.78 and 8.16) was suggested by the correlations observed in the 1H−1H COSY. The HMBC correlations shown in Figure 3 justified the assignments of the seven quaternary carbons. The HMBC correlations and NOE experiments revealed that an acridine ring moiety existed with a hydroxymethyl group on C-2.22−24 The phenolic moiety was confirmed by the presence of an additional exchangeable proton signal at δH 9.80 (OH). These data led to the structure elucidation of inubosin A. The molecular formula of inubosin B (2) was the same as 1. Comparison of 1H and 13C NMR data obtained for 1 and 2 confirmed their similarity except for the position of a phenolic moiety. Four proton signals (δ 7.60, 7.82, 8.14, and 8.19) were linked to the contiguous carbon atoms by coupling constants B
DOI: 10.1021/np5006218 J. Nat. Prod. XXXX, XXX, XXX−XXX
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Figure 4. Ngn2 promoter activities of inubosins (white bars) and cytotoxicity against the assay cells (solid bars). The assay cells were treated with compounds for 24 h. Baicalin (100 μM) was used as a positive control. Assays were performed in 0.1% DMSO (n = 3). Error bars represent SD. of liquid ISP2 medium and incubated at 27 °C for 7 days with reciprocal shaking. Extraction and Isolation. The culture broth (24 L) was harvested and centrifuged to separate the mycelium and the supernatant. The supernatant was subjected to HP20 column chromatography (50 × 400 mm) and eluted with gradient mixtures of H2O−MeOH−acetone (1/0/0−0/0/1) to give three fractions (frs. 1A−1C). All three fractions showed Ngn2 promoter activity. Fr. 1C (1.2 g) was subjected to Sephadex LH20 column chromatography (30 × 250 mm) and eluted with MeOH to give three fractions (frs. 4A−4C). Active fr. 4C (86 mg) was subjected to ODS column chromatography (20 × 300 mm) and eluted with gradient mixtures of H2O−MeOH (1/0−0/1) to give five fractions (frs. 5A−5E). Active fr. 5B (12 mg) was subjected to reversed-phase HPLC [YMC-Pack ODS-AM, 10 × 250 mm; eluent, H2O−MeOH (50:50); flow rate, 2.0 mL/min] to give six fractions (frs. 6A−6F) and to afford 1 (1.6 mg, tR 31 min) and 3 (2.4 mg, tR 36 min). Active fr. 6C (1.8 mg) was subjected to reversed-phase HPLC [Develosil ODS-MG5, 10 × 250 mm; eluent, H2O−MeOH (50:50); flow rate, 2.0 mL/min] to afford 2 (0.9 mg, tR 44 min). For an increase in each compound, other culture broth (21 L) was harvested and centrifuged to separate the mycelium and the supernatant. The supernatant was subjected to HP20 column chromatography (50 × 400 mm) and eluted with gradient mixtures of H2O−MeOH−acetone (1/0/0−0/0/1) to give three fractions (frs. 7A−7C). Active fr. 7C (1.6 g) was subjected to Sephadex LH20 column chromatography (30 × 250 mm) and eluted with MeOH to give three fractions (frs. 8A−8C). Active fr. 8C (127 mg) was subjected to ODS column chromatography (20 × 300 mm) and eluted with gradient mixtures of H2O−MeOH (1/0−0/1) to give five fractions (frs. 9A−9E). Active fr. 9D (22 mg) was subjected to reversed-phase HPLC [Develosil C30-UG5, 10 × 250 mm; eluent, H2O−MeOH (50:50); flow rate, 2.0 mL/min] to give four fractions (frs. 12A−12D) and to afford 3 (15 mg, tR 60 min). Active fr. 12B (4.2 mg) was subjected to reversed-phase HPLC (Develosil ODS-MG5, 10 × 250 mm; eluent, H2O−MeOH (50:50); flow rate, 2.0 mL/min) to afford 2 (2.7 mg, tR 44 min). Inubosin A (1): UV (MeOH) λmax (log ε) 376 (2.8), 341 (2.9), 259 (4.0) nm; IR (ATR) νmax 3358, 1490, 1361, 1239 cm−1; see Table 1 for NMR data; HRESIMS m/z 224.0735 [M − H]− (calcd for C14H10NO2, 224.0711, Δ +2.4 mmu). Inubosin B (2): UV (MeOH) λmax (log ε) 378 (2.7), 358 (2.9), 260 (4.1) nm; IR (ATR) νmax 3333, 1472, 1369, 1230 cm−1; see Table 1 for NMR data; HRESIMS m/z 224.0696 [M − H]− (calcd for C14H10NO2, 224.0711, Δ −1.5 mmu). Inubosin C (3): UV (MeOH) λmax (log ε) 398 (2.9), 262 (4.2) nm; IR (ATR) νmax 3385, 1472, 1352, 1230 cm−1; see Table 1 for NMR data; HRESIMS m/z 240.0656 [M − H]− (calcd for C14H10NO3, 240.0660, Δ −0.4 mmu).
Figure 5. Effects of compounds 1 and 2 (20 μM) on gene expression in C17.2 cells. Baicalin was used as a positive control (20 μM). The gene expression of pro-neural transcriptional factors (Ngn2, Ngn1, NeuroD2) and a pro-neural neurotrophic growth factor (NT3) was investigated with real-time RT-PCR. The mRNA level was determined after cells were treated with the compounds for 4 days. The mRNA level was normalized to the level of GAPDH. Error bars represent SD.
Ngn2 promoter activity. A precise mechanistic study and further chemical transformation to obtain more active compounds are in progress.
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EXPERIMENTAL SECTION
General Experimental Procedures. The NMR data were obtained on JEOL ECP400, A400, and A500 spectrometers. ESIMS spectra were measured with a JEOL JMS-T100LP. IR spectra were recorded on an ATR in a Jasco FT-IR 230 spectrophotometer. UV spectra were recorded on a Shimadzu UV-mini-1240 spectrometer. Luciferase assay, cell culture, RNA isolation, and real-time RT-PCR analysis were performed as described before.20 Microbial Strain. Streptomyces sp. IFM 11440 was isolated from a soil sample collected from Choshi City in Chiba Prefecture, Japan. The identification was carried out by Professor Tohru Gonoi at the Medical Mycology Research Center, Chiba University, where a voucher specimen is deposited with code IFM 11440. The gene sequence is shown in the Supporting Information. Fermentation. Spores of Streptomyces sp. IFM 11440 growing on solid ISP2 medium (1% malt extract, 0.4% yeast extract, and 0.4% glucose) were inoculated into Sakaguchi flasks each containing 100 mL of liquid ISP2 medium and then incubated at 27 °C for 3 days with reciprocal shaking to produce a seed culture. The seed culture (10 mL) was then inoculated into 32 flasks (3 L) each containing 750 mL C
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(19) Li, M.; Tsang, K.-S.; Choi, S.-T.; Li, K.; Shaw, P.-C.; Lau, K.-F. ChemBioChem 2011, 12, 449−456. (20) Arai, M. A.; Koryudzu, K.; Koyano, T.; Kowithayakorn, T.; Ishibashi, M. Mol. Biosyst. 2013, 9, 2489−2497. (21) Larsson, R.; Kristensen, J.; Sandberg, C.; Nygren, P. Int. J. Cancer 1992, 50, 177−185. This assay is based on the measurement of fluorescence generated from cellular hydrolysis of fluorescein diacetate to fluorescein by esterase in living cells. (22) Roubaud, G.; Faure, R.; Galy, J.-P. Magn. Reson. Chem. 2003, 41, 549−553. (23) Bontemps, N.; Bry, D.; López-Legentil, S.; Simon-Levert, A.; Long, C.; Banaigs, B. J. Nat. Prod. 2010, 73, 1044−1048. (24) Bontemps, N.; Gattacceca, F.; Long, C.; Thomas, O. P.; Banaigs, B. J. Nat. Prod. 2013, 76, 1801−1805. (25) Snyder, E. Y.; Deitcher, D. L.; Walsh, C.; Arnold-Aldea, S.; Hartwieg, E. A.; Cepko, C. L. Cell 1992, 68, 33−51. (26) Hindley, C.; Ali, F.; McDowell, G.; Cheng, K.; Jones, A.; Guillemot, F.; Philpott, A. Development 2012, 139, 1718−1723. (27) Takahashi, J.; Palmer, T. D.; Gage, F. H. J. Neurobiol. 1999, 38, 65−81.
ASSOCIATED CONTENT
S Supporting Information *
This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Phone/Fax: + 81-43-2262924. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank Prof. T. Gonoi (Medical Mycology Research Center, Chiba University) for the identification of Streptomyces sp. IFM11440. We are very grateful to Prof. R. Kageyama and Prof. T. Ohtsuka for the kind provision of plasmids and discussions. This study was supported by a Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (JSPS), a Grant-in-Aid for Scientific Research on Innovative Areas “Chemical Biology of Natural Products” from the Ministry of Education, Culture, Sports, Science and Technology, Japan (MEXT), the Asian Core Program (JSPS), Hayashi Memorial Foundation for Female Natural Scientists, the Naito Foundation, and the Tokyo Biochemical Research Foundation.
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REFERENCES
(1) Gage, F. H. Science 2000, 287, 1433−1438. (2) Alvarez-Buylla, A.; Garcia-Verdugo, J. M. J. Neurosci. 2002, 22, 629−634. (3) Johansson, C. B.; Momma, S.; Clarke, D. L.; Risling, M.; Lendahl, U.; Frisen, J. Cell 1999, 96, 25−34. (4) Kim, D. E.; Schellingerhout, D.; Ishii, K.; Shah, K.; Weissleder, R. Stroke 2004, 35, 952−957. (5) Yamashita, T.; Ninomiya, M.; Hernández Acosta, P.; GarcíaVerdugo, J. M.; Sunabori, T.; Sakaguchi, M.; Adachi, K.; Kojima, T.; Hirota, Y.; Kawase, T.; Araki, N.; Abe, K.; Okano, H.; Sawamoto, K. J. Neurosci. 2006, 26, 6627−6636. (6) Imayoshi, I.; Kageyama, R. Neuron 2014, 82, 9−23. (7) Imayoshi, I.; Isomura, A.; Harima, Y.; Kawaguchi, K.; Kori, H.; Miyachi, H.; Fujiwara, T.; Ishidate, F.; Kageyama, R. Science 2013, 342, 1203−1208. (8) Powell, L. M.; Jarman, A. P. Curr. Opin. Genet. Dev. 2008, 18, 411−417. (9) Bertrand, N.; Castro, D. S.; Guillemot, F. Nat. Rev. Neurosci. 2002, 3, 517−530. (10) Hsieh, J.; Nakashima, K.; Kuwabara, T.; Mejia, E.; Gage, F. H. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 16659−16664. (11) Warashina, M.; Min, K. H.; Kuwabara, T.; Huynh, A.; Gage, F. H.; Schultz, P. G.; Ding, S. Angew. Chem., Int. Ed. 2006, 45, 591−593. (12) Zhu, S.; Wurdak, H.; Wang, J.; Lyssiotis, C. A.; Peters, E. C.; Cho, C. Y.; Wu, X.; Schultz, P. G. Cell Stem Cell 2009, 4, 416−426. (13) Wurdak, H.; Zhu, S.; Min, K. H.; Aimone, L.; Lairson, L. L.; Watson, J.; Chopiuk, G.; Demas, J.; Charette, B.; Halder, R.; Weerapana, E.; Cravatt, B. F.; Cline, H. T.; Peters, E. C.; Zhang, J.; Walker, J. R.; Wu, C.; Chang, J.; Tuntland, T.; Cho, C. Y.; Schultz, P. G. Proc. Natl. Acad. Sci. U.S.A. 2010, 107, 16542−16547. (14) Kim, G.-H.; Halder, D.; Park, J.; Namkung, W.; Shin, I. Angew. Chem., Int. Ed. 2014, 126, 9425−9428. (15) Jin, G.; Tan, X.; Tian, M.; Qin, J.; Zhu, H.; Huang, Z.; Xu, H. Neurosci. Lett. 2005, 386, 105−110. (16) Liu, J. W.; Tian, S. J.; de Barry, J.; Luu, B. J. Nat. Prod. 2007, 70, 1329−1334. (17) Maden, M. Nat. Rev. Neurosci. 2007, 8, 755−765. (18) Katakura, M.; Hashimoto, M.; Shahdat, H. M.; Gamoh, S.; Okui, T.; Matsuzaki, K.; Shido, O. Neuroscience 2009, 160, 651−660. D
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