Hes1-Binding Compounds Isolated by Target Protein Oriented Natural

Feb 13, 2017 - Hes1-Binding Compounds Isolated by Target Protein Oriented Natural Products Isolation (TPO-NAPI). Midori A. Arai† , Mitsuha Tanaka†...
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Hes1-Binding Compounds Isolated by Target Protein Oriented Natural Products Isolation (TPO-NAPI) Midori A. Arai,*,† Mitsuha Tanaka,† Kana Tanouchi,† Naoki Ishikawa,† Firoj Ahmed,‡ Samir K. Sadhu,§ and Masami Ishibashi*,† †

Graduate School of Pharmaceutical Sciences, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8675, Japan Department of Pharmaceutical Chemistry, University of Dhaka, Dhaka1000, Bangladesh § Pharmacy Discipline, Khulna University, Khulna 9208, Bangladesh ‡

S Supporting Information *

ABSTRACT: Hairy and enhancer of split 1 (Hes1) is a transcription factor that acts in neural stem cells to inhibit differentiation. We recently developed target protein oriented natural products isolation (TPO-NAPI) using Hes1-immobilized beads to identify activators of neural stem cells. Isomicromonolactam (1), staurosporin (2), and linarin (3) were isolated as Hes1-binding compounds using the TPONAPI method. Of these, compound 1 enhanced neural stem cell differentiation. Using truncated Hes1 proteins, the binding region of Hes1 for 1 was estimated to be in the C-terminal half that includes a TLE/Grg binding site. The differentiation-promoting activity of inohanamine (4) is also reported.

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eural stem cells can differentiate into neurons, astrocytes, and oligodendrocytes and were initially discovered in mouse1 and adult human brain (dentate gyrus,2 subventicular zone3). Two types of transcription factors are included in the family of basic helix loop helix (bHLH) factors:4 activator-type bHLH factors, including Neurogenin2 and NeuroD, and repressor-type bHLH factors, including hairy and enhancer split 1 (Hes1) and Hes5. Activator-type bHLH factors activate the differentiation of neural stem cells, whereas repressor-type bHLH factors inhibit the expression of activator-type bHLH factors by binding to their promoter region. Therefore, the identification of a Hes1 inhibitor would provide a potential candidate drug for treating neural diseases. To find such a candidate from natural resources, we recently developed the Hes1 bead HPLC method, based on the idea of target protein oriented natural products isolation (TPO-NAPI).5,6 After generating Hes1-immobilized Sepharose beads, the Hes1 beads were mixed with natural extracts from plants and actinomyces. We recently isolated three flavonoid glycosides, αmangostine, a macrolactam BE-14106, and alkaloid inohanamine using this method and reported their effect on neural stem cell differentiation activity.5 Here, we describe the isolation of a new natural product and two known compounds that exhibit Hes1-binding activity and also report the neural stem cell activation activity of a previously reported alkaloid inohanamine.5 A schematic of the screening method for Hes1-binding natural products is shown in Figure 1. Glutathione-S-transferase (GST)-fused Hes1 was immobilized on beads (GST-Hes1 beads). These beads were mixed and incubated with natural product extracts. Crude natural product extracts provided many peaks upon HPLC analysis corresponding to the various natural © 2017 American Chemical Society and American Society of Pharmacognosy

Figure 1. Target protein-oriented natural product isolation (TPONAPI). (a) HPLC profile of a natural product extract; (b) incubation of the extract with Hes1 beads; (c) washing the beads and release of compounds from Hes1; (d) detection of the Hes1-binding natural product; (e) representation of the Hes1-glutathione Sepharose beads.

product components (Figure 1a). Unbound natural product compounds were washed from the beads; then natural products bound to Hes1 were released by the addition of EtOH, followed by heating and analysis of the released compounds by HPLC (Figure 1d). The use of information such as retention times and UV absorption patterns allowed facile isolation of the desired natural product components. The most promising component was chosen by identifying nonspecifically binding compounds using GST-immobilized beads at a concentration comparable to that of GST-Hes1 protein. Received: November 19, 2016 Published: February 13, 2017 538

DOI: 10.1021/acs.jnatprod.6b01072 J. Nat. Prod. 2017, 80, 538−543

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Figure 2. HPLC chromatograms of Micromonospora sp. IFM 11769 extract (left) and Hes1-binding natural products after screening (right). The amount of protein on the beads was approximately 3.6 nmol for GST-Hes1 beads and 3.8 nmol for GST beads. Each mixture of beads (bed volume 100 μL) and extract (125 μg in EtOH, 25 μL) was incubated at 4 °C for 2 h. After washing, the bound natural products were dissociated from Hes1 by the addition of 70% EtOH, followed by heating (100 °C, 3 min).

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RESULTS AND DISCUSSION

has the E in a C14/C15 double bond and the Z in a C22/C23 double bond, whereas isomicromonolactam (1) has the Z in a C14/C15 double bond and the E in a C22/C23 double bond. We also obtained extracts of CKK1184 and Cirsium arvense, a plant collected in Bangladesh, and found that they contained Hes1-binding compounds. Because the information on retention time of Hes1-binding compounds was obtained by this method, compounds were isolated by using peak-guided fractionation. A bisindole alkaloid, staurosporine (2),8−11 was isolated from the extract of CKK1184, and a flavonoid glycoside, linarine (3),12 was obtained from the extract of Cirsium arvense.

Our natural products extract libraries of tropical plants and actinomycete strains were screened using the Hes1 bead assay method. The results from Micromonospora sp. IFM 11769, which was isolated from a soil sample collected at Odaibakaihin Park, Tokyo, Japan, are shown in Figure 2; other HPLC results of hit extracts are provided in the Supporting Information. The left-hand panel shows the HPLC profile of the EtOAc extract of Micromonospora sp. IFM 11769, and many UV-absorbing peaks are evident. After mixing with GST-Hes1 beads, followed by washing and release of the bound compounds as described above, several peaks were observed in the supernatant. GST beads (control) were also incubated with the EtOAc extract of Micromonospora sp. After comparison of the two HPLC profiles, one peak was selected as a compound that binds to Hes1 protein, and thus a new natural product, isomicromonolactam (1), was isolated. Isomicromonolactam (1) has the same molecular formula as that of micromonolactam,7 C28H39NO5. Isomicromonolactam (1) was obtained as a light yellow solid, and its molecular formula C28H39NO5 was determined by HRESITOFMS. The UV absorption spectrum (λmax 302, 342, 360 nm) was indicative of a polyene moiety. The 13C NMR data of 1 indicated 28 carbons, including 15 olefinic methines, one carbonyl, two methylenes, and three methyl carbons (Table 1). COSY, HMBC, and NOESY correlations indicated a triene system conjugated with a carbonyl. The coupling constants for protons H-2 through H-7 were consistent with the E geometry for all three double bonds (Figure 3). COSY and NOESY correlations further extended the structure from H-7 through to the H-13 oxymethine and were indicative of four oxymethines and a methylene. The remaining olefinic resonances and COSY and HMBC correlations showed the existence of another polyene portion, from C-14 to C-23. Coupling constants revealed their stereochemistry, which was also elucidated by NOESY correlations. The differences between isomicromonolactam (1) and micromonolactam were the geometry of two double bonds. Micromonolactam

With these isolated Hes1-binding natural compounds in hand, the ability of the compounds to accelerate neural stem cell differentiation was evaluated (Figure 4). Multipotent 539

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mouse neural stem cells (MEB5)13 were treated with DMSO (control), valproic acid (100 μM) (positive control),14,15 or the individual isolated compounds for 4 days. Confocal images of the differentiated neural cells were obtained after immunostaining class III β-tubulin (Tuj1) in the neurons, and the number of neurons was calculated. Only compound 1 and inohanamine (4), previously isolated as a Hes1-binding compound,5 increased the number of neurons at a lower concentration (10 μM) compared to the concentration required for a comparable increase by the positive control (valproic acid; 100 μM). The percentage of neurons differentiated following treatment with 1 and 4 was 30% and 35%, respectively, a 17% and 34% increase compared to that of the DMSO control. To identify the binding region of isomicromonolactam (1) to Hes1, truncated Hes1 proteins were synthesized as previously reported (Figure 5A).5 Isomicromonolactam (1) was incubated with each protein (Full, 1−95 aa, 47−156 aa, 47−281 aa, 151− 281 aa) immobilized on beads. Binding compound (1) was released by the addition of EtOH to the washed beads. The amounts that had bound to the beads were compared by UV absorption (Figure 5B). The truncated Hes1 proteins Part III (47−281 aa) and Part IV (151−281 aa) showed significant binding to isomicromonolactam (1). These truncated Hes1 proteins contain the domain that binds TLE/Grg, a corepressor for transcription. On the other hand, Part I (1−95 aa) and Part II (47−156 aa) bound weakly to isomicromonolactam (1). The binding abilities of these truncated proteins predicted from the bead assay results are shown in Figure 5C, and the isomicromonolactam (1) binding site was predicted to be in the C-terminal half of the protein. The binding of 1 to the Cterminal half might prevent the binding of TLE/Grg required to inhibit the transcription of activated bHLH factors, thus preventing Hes1 inhibition. This could be one mechanism by which 1 activates the differentiation of neural stem cells. In conclusion, we report here the isolation of three compounds, including one new natural product, by TPONAPI using Hes1-immobilized beads. This method would be useful for isolating natural products that bind to target proteins.

Table 1. 1H and 13C NMR Spectroscopic Data of 1 in DMSO-d6 position

δ Ca

1 2 3 4 5 6 7 8 9 10 11 12

165.8 125.3 138.0 128.8 138.0 128.1 142.9 37.4 74.0 74.0 71.1 39.5

13 14 15 16 17 18 19 20 21 22 23 24

67.7 137.3 126.7 123.9 136.6 135.4 130.9 132.4 128.1 134.5 130.2 40.2

25 26 27 28 NH 9-OH 10-OH 11-OH 13-OH

44.9 21.2 11.4 12.1

δH (J in Hz) 5.75 6.72 6.18 6.29 6.06 5.89 2.61 3.40 3.13 4.03 1.72 1.36 4.71 5.46 5.93 6.45 6.22

d (15.5) dd (15.5, 11.1) m dd (10.4, 14.1) dd (14.9, 10.4) dd (14.9, 6.5) m m dd (7.8, 7.2) m dd (13.4, 9.6) ddd (13.4, 9.5, 4.5) m dd (10.8, 7.8) dd (11.0, 10.8) dd (14.6, 11.0) d (14.6)

6.06 6.15 6.33 6.07 5.54 2.26 2.11 3.70 1.16 0.92 1.76 7.52 4.45 4.42 4.29 4.73

d (10.8) dd (14.3, 10.8) dd (14.3, 11.3) dd (14.8, 11.3) m ddd (10.8, 4.6, 3.9) ddd (11.4, 10.9, 10.8) m d (6.6) d (7.2) s d (9.0) d (4.8) d (7.8) m s

a13

C chemical shifts were determined from HMQC data.

Figure 3. (A) Key 1H−1H COSY and HMBC data of 1; (B) key coupling constants of 1; (C) key NOESY correlations of 1. 540

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Figure 4. MEB5 differentiation-promoting activity. The effects on neurons were quantified. MEB5 cells were treated with DMSO (dimethyl sulfoxide, negative control), VPA (valproic acid, positive control; 100 μM), isomicromonolactam (1) (10 μM), or inohanamine (4) (10 μM) for 4 days. The cells were then immunostained with Tuj1 (green) for neurons and TO-PRO-3 (blue) for nuclei.

Figure 5. Binding ability of compound 1 to Hes1-truncated proteins. The concentrations of all GST-Hes1 proteins were approximately 3.6 nmol. (A) Schematic showing the synthesized full and truncated Hes1 proteins. (B) Results of the amount of compound 1 bound to each type of protein bead. (a) GST-Hes1 (1−281 aa) beads, (b) GST-Hes1 (1−95 aa) beads, (c) GST-Hes1 (47−156 aa) beads, (d) GST-Hes1 (47−281 aa) beads, (e) GST-Hes1 (151−281 aa) beads. The mass of bound compound was calculated from the area of the product peak. (C) Binding ability of compound 1 predicted from the results of the bead assay.

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was carried out on a Shimadzu LC-20AT pump that was equipped with a Shimadzu SPD-M20A photodiode array detector and a Shimadzu LCMS-2020. Column chromatography was performed using silica gel PSQ100B, Chromatorex DIOL (Fuji Silysia Chemical Ltd., Kasugai, Japan), and silica gel 60N (Kanto Chemical Co., Inc., Tokyo, Japan). Analytical HPLC was performed using CAPCELL PAK C18 MGII (Shiseido Co., Ltd., Yokohama, Japan). Preparative HPLC was performed using Develosil C30-UG-5 (Nomura Chemical Co., Ltd., Seto, Japan).

EXPERIMENTAL SECTION

General Experimental Procedures. NMR spectra were recorded on JEOL ECP600 and ECZ600 spectrometers in a deuterated solvent whose chemical shift was taken as an internal standard. Electrospray ionization mass spectra (ESIMS) were obtained using a JEOL JMST100LP and a Shimadzu LCMS-2020. HPLC was carried out on a Waters 600 pump that was equipped with a Waters 2996 photodiode array detector or a Shimadzu LC-20AD pump that was equipped with a Shimadzu SPD-M10AVP photodiode array detector. LC-MS analysis 541

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Microbial Strains. Micromonospora sp. IFM 11769 and CKK1184 were isolated from soil samples collected at Odaibakaihin Park, Tokyo, Japan, and at Onjuku Beach, Chiba City, Japan, respectively. Micromonospora sp. was identified by Professor Tohru Gonoi (Medical Mycology Research Center, Chiba University, Japan), and a voucher specimen was deposited under the code IFM 11769. Plant Materials. The aerial part of Cirsium arvense (Asteraceae) was obtained in Bangladesh. A specimen (KKB314) was deposited at the Department of Natural Products Chemistry, Graduate School of Pharmaceutical Sciences, Chiba University, Japan. Expression and Purification of Recombinant GST-Fused Proteins. E. coli strain JM109 (Nippon Gene Co., Ltd., Tokyo, Japan) serves as a host for pGEX-ratHes13−281, pGEX-ratHes11−95, pGEXratHes147−156, pGEX-ratHes147−281, pGEX-ratHes1151−281, and pGEX6P-1. An overnight plateau phase culture of JM109 was inoculated into fresh LB medium (Invitrogen) containing 100 mg L−1 ampicillin. Cells were grown at 37 °C to a density of 0.6 (OD600), and protein synthesis was induced by addition of 0.1 mM IPTG (isopropyl-1-thio-β-Dgalactopyranoside) followed by incubation for an additional 4−8 h at 18 °C. The cells were harvested by centrifugation and lysed by sonication. After incubation with 1% Triton X-100 for 30 min at 4 °C, the lysate was centrifuged at 6000 rpm for 10 min at 4 °C. The resulting supernatant was added to prewashed glutathione Sepharose 4B and gently mixed for 1 h at 4 °C. The beads obtained after centrifugation (2000 rpm, 5 min, at 4 °C) were washed with phosphate-buffered saline (PBS) or WE buffer (20 mM Tris-HCl, pH 7.5, 2 mM MgCl2, and 0.1 mM DTT). The recombinant proteins were eluted with 50 mM glutathione buffer (50 mM Tris-HCl, pH 8.8) and then dialyzed against PBS or NET buffer using Slide-A-Lyzer dialysis cassette (Thermo). The GST was cleaved from recombinant protein by PreScission protease (GE Healthcare) for 4 h at 4 °C in cleavage buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, and 1 mM DTT), then dialyzed against PBS buffer. The protein concentration was determined by a Micro BCA protein assay kit (Thermo). The recombinant proteins were estimated to be greater than 90% pure by SDS-PAGE. Typical Screening Procedure. A PBS solution of GST-Hes1 (200 μg, ca. 3.6 nmol) was added to glutathione Sepharose 4B beads (bed volume 100 μL, GE Healthcare), and this was mixed at 4 °C for 1 h by a rotated mixer. The GST-Hes1 beads were washed five times by NET buffer (20 mM Tris-HCl, pH 7.5, 200 mM NaCl, 1 mM EDTA). The beads were suspended in NET buffer (250 μL). An extract of natural resources (125 μg in EtOH, 25 μL) was added to the above freshly prepared GST-Hes1 beads (bed volume 100 μL), and the mixture was gently mixed for 2 h at 4 °C by a rotated mixer. The beads were washed by a rotated mixer at 4 °C for 10 min three times with NET-N buffer (NET buffer containing 0.05% Nonidet P-40, 500 μL). To the beads was added 70% EtOH (150 μL), and the suspension was heated at 100 °C for 3 min. After centrifugation (2000 rpm, 4 °C, 1 min), the beads were gathered and the supernatant was centrifuged at 15 000 rpm for 15 min. One-third of the supernatant was analyzed by HPLC. The control GST beads were also prepared in the same procedure as the GST-Hes1 beads. A PBS solution of GST (100 μg, ca. 3.8 nmol) was added to prewashed glutathione Sepharose 4B beads (bed volume 100 μL, GE Healthcare). When there is the obvious difference in the peak intensity between the results of GST-Hes1 beads and GST beads (control), such extracts were obtained as “hit” extracts that had the Hes1-binding natural products. Fermentation, Extraction, and Isolation from Actinomycetes. Spores of Micromonospora sp. IFM 11769 growing on solid Waksman medium were inoculated into 2 × 500 mL Sakaguchi flasks each containing 100 mL of liquid Waksman medium and incubated at 27 °C for 5 days with reciprocal shaking at 120 rpm to produce the seed culture. The seed culture (20 mL) was inoculated into each of 6 × 3 L flasks containing 750 mL of liquid Waksman medium and incubated at 27 °C for 5 days with reciprocal shaking at 120 rpm. Liquid Waksman medium was composed of 2% glucose, 0.5% peptone, 0.5% meat extract, 0.3% yeast extract, 0.5% NaCl, and 0.3% CaCO3; 1.5% agar was added for solidification. Next, 4.5 L of acetone was added to the culture broth, and the cultures were further incubated for

4 h to kill the Micromonospora and to extract compounds from the mycelia. The broth was filtered and concentrated in vacuo to 4.5 L. The crude aqueous solution was then extracted with EtOAc (4.5 L × 3). The mycelium was extracted with MeOH and EtOAc. The MeOHsoluble fraction of the mycelium (548.8 mg) and the EtOAc-soluble fraction of the mycelium (29.8 mg) were combined and separated by chromatography on a Develosil ODS-MG-5 (10 × 250 mm) column and eluted with 60% MeOH to afford compound 1 (1.4 mg). Compound 1 is reactive, and thus the isolation was carried out essentially under dark conditions. Spores of CKK1184 growing on solid Waksman medium were inoculated into 2 × 500 mL Sakaguchi flasks each containing 100 mL of liquid Waksman medium and incubated at 27 °C for 5 days with reciprocal shaking at 120 rpm to produce the seed culture. The seed culture (20 mL) was then inoculated into each of 10 × 3 L flasks containing 750 mL of liquid Waksman medium and incubated at 27 °C for 5 days with reciprocal shaking at 120 rpm to obtain 7.5 L of fermentation broth. Next, 7.5 L of acetone was added to the culture broth, and the culture was further incubated for 3 h to extract the active compounds from the mycelium. The broth was filtered and concentrated in vacuo to 7.5 L; then the crude aqueous solution was extracted with EtOAc (7.5 L × 3). After removal of the EtOAc, the extract was suspended in H2O−MeOH (1:9, 300 mL) and partitioned with hexane, EtOAc, and BuOH (60 mL × 3). The EtOAc-soluble fraction (347.1 mg) was subjected to silica gel column chromatography (38 × 290 mm) and eluted with gradient mixtures of CHCl3− MeOH (1:0−0:1) to give 14 fractions (frs. 1A−1N). Fr. 1C (18.7 mg) was subjected to DIOL silica gel column chromatography (13 × 410 mm) and eluted with gradient mixtures of EtOAc−MeOH (1:0−0:1) to afford compound 2 (3.2 mg). Extraction of Cirsium arvense. The air-dried aerial parts of C. arvense (340 g) were extracted with MeOH overnight at room temperature (RT). The MeOH extract of C. arvense (14.8 g) was subjected to Diaion HP-20 column chromatography (85 × 230 mm) and eluted with gradient mixtures of MeOH−acetone (1:0−0:1) to give two fractions (frs. 1A and 1B). Fr. 1A (10.6 g) was suspended in H2O−MeOH (90:10, 300 mL) and partitioned with hexane (300 mL × 3), EtOAc (300 mL × 3), and nBuOH (300 mL × 3). The nBuOHsoluble fraction (1.76 g) was subjected to silica gel column chromatography (50 × 260 mm) and eluted with gradient mixtures of CHCl3−MeOH (1:0−0:1) to give 13 fractions (frs. 2A−2M). Fr. 2F (126 mg) was suspended with MeOH and precipitated (3A; 34.8 mg, 3). Cell Culture. Mouse neural stem cells (MEB5) were purchased from the Health Science Research Resources Bank (HSRRB). MEB5 cells were cultured and propagated as floating neurospheres in “proliferation medium” (NeuroClut basal medium (mouse, VERITAS) supplemented with 10% NSC proliferation supplements (mouse, VERITAS)). Cells were transferred to “differentiation medium” (NeuroCult Basal Medium (mouse) supplemented with 10% NSC proliferation supplements (mouse, VERITAS)). All cultures were maintained in a humidified incubator at 37 °C in 5% CO2/95% air. Differentiation Assay. Floating neurospheres of MEB5 cells were dissociated using NeuroCult chemical dissociation kit (STEMCELL) and plated on glass coverslips coated with poly-L-lysine, fibronectin, and laminine in 24-well plates in proliferation medium at a density of 2 × 104 cells/well. After 12 h of incubation, the cells were washed with NeuroCult basal medium (mouse), then incubated in differentiation medium with compounds at each concentration for 4 days. MEB5 cells were fixed with 4% paraformaldehyde in PBS for 20 min at RT and washed twice with PBS containing 1% bovine serum albumin (BSA). The fixed cells were permeablilized and blocked with 0.3% Triton X100 and 10% BSA in PBS for 45 min at RT and incubated overnight at 4 °C with primary antibodies. After washing with PBS containing 1% BSA, the cells were incubated at RT for 1 h with secondary antibodies that were conjugated with fluorescent dyes. After washing, cells were incubated with 200 μg/mL RNase (Invitrogen) in PBS containing 1% BSA and 0.1% Triton X-100 for 1 h at 37 °C. After washing with PBS, cells were treated with 3 μM TO-PRO-3 (Invitrogen) for 10 min (for nuclei staining) at RT in the dark. Finally, the cells were washed with 542

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PBS and mounted with ProLong Gold antifade reagent with DAPI (Invitrogen). Fluorescence was detected and photographed at RT with a LSM700 confocal laser scaning microscope (Carl Zeiss). A total of nine pictures were taken per well, and the assays were carried out in three individual wells. The analysis was performed by using antibodies to mouse anti-βIII-tubulin (neuron marker, 1/400, R&D) and rabbit anti-GFAP (astrocyte marker, 1/400, VERITAS) as primary antibodies and Alexa Fluor 488 goat anti-mouse IgG (1/400, Invitrogen) and Alexa Fluor 555 goat anti-rabbit IgG (1/200, Invitrogen) as secondary antibodies. Cell count was conducted using ImageJ software. Isomicromonolactam (1) Binding Region in Hes1. The binding region of compound 1 in Hes1 was predicted using the Hes1 bead assay method, truncated proteins of Hes1, and the same procedure as described for screening. Briefly, compound 1 (12.5 nmol in DMSO) was mixed and incubated with full and truncated Hes1 bound to beads (approximately 3.6 nmol each). The quantity of 1 bound to each protein was measured and calculated from the peak area of 1, and the predicted binding ability was expressed as a ratio to GSTHes1 (1−281 aa, Full) beads. Isomicromonolactam (1): light yellow solid; UV (MeOH) λmax 302, 342, and 360 nm; 1H and 13C NMR, see Table 1; HRESIMS m/z 492.2707 [M + Na]+ (calcd for C28H39NO5Na+, 492.2726).

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(5) Arai, M. A.; Ishikawa, N.; Tanaka, M.; Uemura, K.; Sugimitsu, N.; Koyano, T.; Kowithayakorn, T.; Ishibashi, M. Chem. Sci. 2016, 7, 1514−1520. (6) Arai, M. A.; Kobatake, E.; Koyano, T.; Kowithayakorn, T.; Kato, S.; Ishibashi, M. Chem. - Asian J. 2009, 4, 1802−1808. (7) Skellam, E. J.; Stewart, A. K.; Strangman, W. K.; Wright, J. L. C. J. Antibiot. 2013, 66, 431−441. (8) O̅ mura, S.; Iwai, Y.; Hirano, A.; Nakagawa, A.; Awaya, J.; Tsuchiya, H.; Takahashi, Y.; Masuma, R. J. Antibiot. 1977, 30, 275− 282. (9) Furusaki, A.; Hashiba, N.; Matsumoto, T.; Hirano, A.; Iwai, Y.; O̅ mura, S. J. Chem. Soc., Chem. Commun. 1978, 800−801. (10) Meksuriyen, D.; Cordell, G. A. J. Nat. Prod. 1988, 51, 884−892. (11) Funato, N.; Takayanagi, H.; Konda, Y.; Toda, Y.; Harigaya, Y.; Iwai, Y.; O̅ mura, S. Tetrahedron Lett. 1994, 35, 1251−1254. (12) Quitin, J.; Lewin, G. J. Nat. Prod. 2004, 67, 1624−1627. (13) Nakagaito, Y.; Satoh, M.; Kuno, H.; Iwama, T.; Takeuchi, M.; Hakura, A.; Yoshida, T. In Vitro Cell. Dev. Biol.: Anim. 1998, 34, 585− 592. (14) Hsieh, J.; Nakashima, K.; Kuwabara, T.; Mejia, E.; Gage, F. H. Proc. Natl. Acad. Sci. U. S. A. 2004, 101, 16659−16664. (15) Abematsu, M.; Tsujimura, K.; Yamano, M.; Saito, M.; Kohno, K.; Kohyama, J.; Namihira, M.; Komiya, S.; Nakashima, K. J. Clin. Invest. 2010, 120, 3255−3266.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.6b01072. Information regarding the plasmids, isolation charts, and additional information (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*Tel: +81-43-226-2924. E-mail: [email protected] (M. A. Arai). *Tel: +81-43-226-2923. E-mail: [email protected] (M. Ishibashi). ORCID

Midori A. Arai: 0000-0003-0254-9550 Masami Ishibashi: 0000-0002-2839-1045 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Professor Tohru Gonoi (Medical Mycology Research Center, Chiba University) for the identification of Micromonospora sp. IFM 11769. This study was supported by a Grant-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), and by a Workshop on Chirality at Chiba University (WCCU). This work was inspired by the international and interdisciplinary environments of the JSPS Core-to-Core Program “Asian Chemical Biology Initiative”.

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REFERENCES

(1) Reynolds, B. A.; Weiss, S. Science 1992, 255, 1707−1710. (2) Eriksson, P. S.; Perfilieva, E.; Björk-Eriksson, T.; Alborn, A. M.; Nordborg, C.; Peterson, D. A.; Gage, F. H. Nat. Med. 1998, 4, 1313− 1317. (3) Goldman, S. A. J. Neurobiol. 1998, 36, 267−286. (4) Imayoshi, I.; Kageyama, R. Neuron 2014, 82, 9−23. 543

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