Note pubs.acs.org/jnp
Indoloditerpenes from a Marine-Derived Fungal Strain of Dichotomomyces cejpii with Antagonistic Activity at GPR18 and Cannabinoid Receptors Henrik Harms,† Viktor Rempel,‡ Stefan Kehraus,† Marcel Kaiser,§,⊥ Peter Hufendiek,† Christa E. Müller,*,‡ and Gabriele M. König*,† †
Institute for Pharmaceutical Biology, University of Bonn, Nussallee 6, D-53115 Bonn, Germany Pharma Center Bonn, Pharmaceutical Institute, Pharmaceutical Chemistry I, University of Bonn, D-53121 Bonn, Germany § Swiss Tropical and Public Health Institute, Socinstraße 57, 4002 Basel, Switzerland ⊥ University of Basel, Petersplatz 1, 4003 Basel, Switzerland ‡
S Supporting Information *
ABSTRACT: A marine-derived strain of Dichotomomyces cejpii produces the new compounds emindole SB betamannoside (1) and 27-O-methylasporyzin C (2), as well as the known indoloditerpenes JBIR-03 (3) and emindole SB (4). Indole derivative 1 was found to be a CB2 antagonist, while 2 was identified as the first selective GPR18 antagonist with an indole structure. Compound 4 was found to be a nonselective CB1/CB2 antagonist. The new natural indole derivatives may serve as lead structures for the development of GPR18- and CB receptor-blocking drugs.
T
prostate, and ovarian carcinomas.7,8 GPR55 antagonists are considered as potential drugs for the treatment of cancer and neuropathic pain.7−9 Here we report on novel indole derivatives from a marine fungal source. Our study shows that compounds of fungal origin have the potential to serve as lead structures for the development of novel ligands for GPCRs, for CB receptors and the related orphan receptor GPR18. Compound 1 was obtained from the marine sponge-derived fungus Dichotomomyces cejpii, and its structure was deduced via analysis of spectroscopic data. The UV maximum at 281 nm indicated the presence of an aromatic moiety. IR absorptions at 3347 and 1651 cm−1 pointed toward several hydroxy groups and a heterocyclic bound nitrogen, respectively. The molecular formula of compound 1 was deduced from the results of an accurate mass measurement to be C34H49NO6Na and was supported by 1H and 13C NMR data (Table 1). The 13C NMR and DEPT135 spectra denote the presence of 34 resonances for five methyl groups, eight sp3 methylene groups, and five sp2 methine, eight sp3 methine, and eight quaternary carbons in the molecule. Comparison with literature 1H and 13C NMR data of emindole SB (4)10 revealed that 1 structurally was very similar to 4. However, additional 1H and 13C NMR resonance
he secondary metabolism of fungi gives rise to chemically diverse structures, some of which have proven useful in drug development. Cannabinoid (CB) receptors belong to the G protein-coupled receptor (GPCR) superfamily1 and are divided into two distinct subtypes, designated CB1 and CB2. The CB1 receptor is highly expressed in the central nervous system, but is also present in peripheral tissues, including heart, liver, lungs, and kidneys.2 CB1 activation mediates a variety of physiological responses including analgesia, stimulation of appetite, and psychoactive effects, such as euphoria. The CB2 receptor is mainly present in organs and cells of the immune system including the spleen, tonsils, the thymus, Tlymphocytes, macrophages, and B-cells, and its activation results in analgesic and immunomodulatory effects.2 Several reports have indicated the existence of further CB receptors, and the orphan receptors GPR18 and GPR55 have been proposed as potential candidates. GPR18 is highly expressed in the spleen, the thymus, leukocytes, testes, and the endometrium.3 The physiological role of GPR18 is poorly understood. Studies indicate an involvement of the receptor in endometriosis, regulation of intraocular pressure, and microglial activation.3,4 Potent and selective GPR18 agonists and antagonists are urgently needed as pharmacological tools to further explore the physiological effects of this putative novel CB receptor subtype, which has potential as a novel drug target. The primarily G12,13-coupled GPR55 is mainly expressed in the brain, as well as in osteoclasts and osteoblasts.5,6 In addition, the receptor was shown to be highly expressed in several cancer cell lines, such as glioblastoma, astrocytoma, breast cancer, © 2014 American Chemical Society and American Society of Pharmacognosy
Special Issue: Special Issue in Honor of Otto Sticher Received: October 10, 2013 Published: January 28, 2014 673
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constant between the anomeric carbon and the respective proton (1JCH = 155 Hz; Figure S1.8).12 Hydrolysis and HPLCELSD analysis using a chiral-phase column revealed the sugar to have the D-configuration. The mannose is attached via the CH-7 methine group to the indoloditerpene nucleus, due to a heteronulear long-range coupling from H-1′ to C-7. The absolute configuration of the indoloditerpene scaffold of 1 is considered to be the same as that of the co-occurring compound emindole SB, for which the absolute configuration had been determined recently.11,13 The fungal metabolite 1 is thus an indoloditerpene, substituted with a dimethylallylcontaining side chain and a mannose moiety, for which we suggest the trivial name emindole SB beta-mannoside.
Table 1. NMR Spectroscopic Data of Compounds 1 and 2 in Acetone-d6 (1H, 300 MHz; 13C, 75 MHz) 1 pos.
δC, mult
1 2 3 4 5 6
NH 152.0, C 53.8, C 39.7, C 33.3, CH2 26.8, CH2
7 8 9 10
83.9, 42.0, 40.7, 23.3,
11
25.9, CH2
12 13
49.7, CH 28.0, CH2
14 15 16 17 18 19 20 21 22 23 24
117.7, C 126.0, C 118.6, CH 119.6, CH 120.5, CH 112.5, CH 141.5, C 15.0, CH3 19.4, CH3 17.9, CH3 38.2, CH2
25
22.2, CH2
26 27 28 29 30 1′ 2′ 3′ 4′ 5′
125.8, CH 131.1, C 25.9, CH3 17.8, CH3
6′
63.2, CH2
CH C CH CH2
103.5, CH 72.2, CH 75.5, CH 69.2, CH 77.6, CH
2
δH (J in Hz) 9.80, brs
1.81, m 6a: 2.10, m 6b: 1.86, m 3.62, m 1.81, m 10a: 1.63, m 10b: 1.45, m 11a: 1.75, m 11b: 1.65, m 2.75, m 13a: 2.62, dd (6.2, 13.2) 13b: 2.29, dd (10.6, 13.2)
7.31, 6.92, 6.96, 7.28,
dd (1.8, 7.6) td (7.6, 1.8) td (7.6, 1.8) dd (1.8, 7.6)
1.03, s 1.11, s 0.83, s 24a: 1.50, m 24b: 1.27, m 25a: 2.03, m 25b: 1.93, m 5.13, t (7.0) 1.66, s 1.64, s
δC/N, mult NH 151.9, C 53.7, C 39.9, C 33.3, CH2 28.3, CH2 72.9, 43.1, 41.1, 23.2,
CH C CH CH2
25.9, CH2 49.7, CH 27.9, CH2
117.7, C 125.9, C 118.6, CH 119.5, CH 120.5, CH 112.5, CH 141.5, C 14.8, CH3 19.1, CH3 16.8, CH3 40.6, CH2
126.6, CH 139.2, CH 75.2, C 26.9, CH3 26.3, CH3 50.3, CH3
δH (J in Hz) 9.77, brs
1.80, 1.80, 1.68, 3.52,
m m m m
1.69, m 10a: 1.75, m 10b: 1.46, m 11a: 1.74, m 11b: 1.59, m 2.75, m 13a: 2.61, dd (6.2, 13.2) 13b: 2.28, dd (10.6, 13.2)
7.30, 6.90, 6.95, 7.28,
Chart 1
dd (1.8, 7.7) td (7.7, 1.8) td (7.7, 1.8) dd (1.8, 7.7)
0.98, s 1.10, s 0.84, s 24a: 2.45, dd (6.2, 13.9) 24b: 1.95, dd (8.8, 13.9) 5.66, ddd (6.2, 8.8, 15.7)
The structure of the fungal metabolite 2 was found to be closely related to that of 1. The molecular formula of 2 was deduced as C29H41NO2Na from the results of an accurate mass measurement. The structure of compound 2 differs from that of compound 1 solely by the missing sugar moiety at C-7 and some structural changes in the terpene side chain. The 13C NMR resonance frequency at δC 72.9 for the methine group CH-7 was shifted upfield by 11 ppm as compared to 1 and, thus, verified the presence of an unsubstituted hydroxy group at that position.10 The structure of the terpenoid chain was elucidated by COSY correlations from H2-24 through to H-26 and HMBC correlations between the methyl groups CH3-28 and CH3-29 and CH-26. The configuration of the double bound between C-25 and C-26 was determined as E, based on a coupling constant of JH‑25/H‑26 = 15.7 Hz. Since C-27 was quaternary in 2 and resonated at δC 75.2, the methoxy group in the molecule, which was evident from 1H and 13C NMR resonance frequencies at δH 3.12 and δC 50.3, was clearly located at C-27. Furthermore, the NMR chemical shifts of the terpenoid moiety were identical with those of the 4-methoxy-4methylpent-2-enyl unit in momordicoside K.13 The configuration of 2 was assigned by comparison to 1 and 4. For the fungal metabolite 2 we suggest the name 27-O-methylasporyzin C. Comparison of NMR and MS data with literature values led to the identification of compounds 3 and 4 as the indoloditerpenes JBIR-03 and emindole SB, respectively.10,14 It is remarkable that the optical rotation of JBIR-03 could not be reliably measured in methanol as reported in the literature, [α]24.5D +46.2 (c 0.05, MeOH),14 due to the low solubility in this solvent. Our repeated measurements of JBIR-03 in
5.50, d (15.7) 1.22, s 1.22, s 3.12, s
4.63, s 3.89, brd (3.3) 3.42, brdd (3.3, 9.2) 3.64, m 3.25, ddd (3.3, 5.9, 9.2) 6′a: 3.82, dd (3.3, 12.1) 6′b: 3.69, dd (5.9, 12.1)
frequencies pointed toward a sugar moiety in the molecule. Thus, the methine groups CH-7, CH-1′, CH-2′, CH-3′, CH-4′, and CH-5′ and the methylene group CH2-6′ resonate in the range δH 3.2 to 4.6 and δC 63 to 104, respectively. The COSY spectrum showed correlations for a spin system from H-1′ through to H2-6′ and established a pyranose moiety. Analysis of the coupling constants and NOEs revealed a mannopyranose.11 The anomeric configuration of the mannose was considered as β because of the magnitude of the heteronuclear coupling 674
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Table 2. Biological Evaluation of Compounds 1−4 at Human CB Receptors and Orphan Receptors GPR18 and GPR55 radioligand binding vs [3H]CP55,940
β-arrestin assays
Ki ± SEM (μM)
IC50 (μM)
compd
CB1
CB2
GPR18
emindole SB beta-mannoside (1) 27-O-methylasporyzin C (2) JBIR-03 (3) emindole SB (4)
>10 (30%)c >10 (39%)c 4.40 ± 1.07 7.05 ± 1.04
10.6 ± 1.8 >10 (33%)c 4.76 ± 1.39 2.24 ± 0.30
>10 (15%)a 13.4 ± 1.3 9.91 ± 2.59 >10 (37%)a
GPR55 >10 >10 >10 >10
(13%)b (31%)b (0%)b (12%)b
% inhibition of THC (10 μM)-mediated β-arrestin recruitment. b% inhibition of LPI (1 μM)-mediated β-arrestin recruitment. c% inhibition of [ H]CP55,940 binding.
a
3
Figure 1. Concentration−inhibition curves. (A) Inhibition of specific [3H]CP55,940 binding by compound 1 at CB1 receptors (Ki CB1 10.6 μM); (B) inhibition of specific [3H]CP55,940 binding by compound 4 at CB1 (▼, Ki 7.05 μM) and CB2 receptors (■, Ki 2.24 μM); (C, D) inhibition of Δ9-THC (7.5 μM)-induced β-arrestin recruitment by 2 (C, IC50 13.4 μM) and 3 (D, IC50 9.91 μM), respectively. Data points represent means ± SEM of three independent experiments, performed in duplicate.
acetonitrile showed negative optical rotation values, similar to other indoloditerpenes, [α]25D −18.1 (c 1.10, CH3CN). Synthetic CB receptor ligands based on an indole scaffold have been described, including the CB1/CB2 agonist (R)-[2,3dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,2,3-de]1,4-benzoxazin-6-yl]-1-naphthalenylmethanone (WIN55,2122) and the CB2 antagonist/inverse agonist 6-iodo-2-methyl-1[2-(4-morpholinyl)ethyl]-1H-indol-3-yl](4-methoxyphenyl)methanone (AM630).15 Therefore, indoloditerpenes 1−4 were investigated in radioligand binding and functional assays (inhibition of forskolin-stimulated cAMP accumulation) at human CB1 and CB2 receptors. In addition we studied indole derivatives 1−4 for their interaction with the CB receptor-like orphan GPCRs GPR18 and GPR55 using β-arrestin translocation assays.16 All examined indole derivatives interacted with one or more of the four investigated receptors (Table 2). Examples for concentration−inhibition curves are shown in Figure 1. None of the compounds showed any significant effect on GPR55 at the highest test concentration of 10 μM. All of the active compounds were characterized as receptor antagonists in functional assays at CB1 and/or CB2 receptors and/or GPR18 (Table 2 and Supporting Information, Table S8). The new fungal metabolite emindole SB beta-mannoside (1), a pentacyclic indole derivative, preferably bound to the CB2 receptor with a Ki value of 10.6 μM. cAMP accumulation assays showed that it was a CB2 antagonist since it did not lead to receptor activation (Table S8). It is surprising that the CB2
receptor accepted the polar sugar moiety attached to the lipophilic core structure in compound 1. One explanation may be that the sugar moiety points toward the extracellular aqueous space of the receptor. In contrast to the majority of known CB receptor ligands, indole derivative 1 shows high water solubility due to the attached mannose. Thus, compound 1 may be a starting point for the development of water-soluble CB2 receptor antagonists with increased affinity. Its aglycone, emindole SB (4), was about 5-fold more potent at the CB2 receptor (Ki 2.24 μM) than mannoside 1 and displayed additional affinity for the CB1 receptor (Ki 7.05 μM). The new pentacyclic indole derivative 27-O-methylasporyzin C (2), which lacks the sugar moiety of 1 and features a different side chain, was found to be an antagonist at the CB-like receptor GPR18 with an IC50 value of 13.4 μM measured in a functional assay versus 7.5 μM tetrahydrocannabinol (THC) (corresponding to its EC80) (Table 2, Figure 1C). Compound 2 showed some selectivity relative to the other investigated receptors, CB1, CB2, and GPR55. So far, selective GPR18 antagonists are unknown. Compound 3, a hexacyclic indole derivative, was almost equipotent at CB1 (Ki 4.40 μM) and CB2 (Ki 4.76 μM) receptors and similarly active at GPR18 (IC50 9.91 μM). At GPR18, the inhibition curves for compounds 2 and 3 went down below basal values (Figure 1C and D), indicating an inverse agonistic mode of action for these two compounds. The herein described emindole SB beta-mannoside (1) is the first CB receptor antagonist from a fungal source with a 675
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fractions. Subfraction 7.6 was further purified by RP C18-HPLC (Nucleodur 100-S 250 mm × 8 mm) using MeOH−H2O (95:5), 2.0 mL min−1. Compound 1 was isolated as a white powder in subfraction 7.6, tR 10 min (6.5 mg). VLC fraction 2 was separated with an NP VLC, eluted stepwise with CH2Cl2−petroleum ether (50:50), up to 100% CH2Cl2, and 100% MeOH, which yielded 15 fractions. Subfraction 2.1 and VLC fraction 1 were separated three times with a solvent/solvent extraction with hexane (100%) and MeOH−H2O (60:40) using a separation funnel. The combined hexane phases were further separated with an NP VLC column and eluted stepwise with CH2Cl2−petroleum ether (20:80), up to 100% CH2Cl2, 100% acetone, and 100% MeOH, which yielded 16 fractions. Subfraction 2.1.7 was further separated with an RP18-SPE cartridge, eluted stepwise with MeOH−H2O (50:50), MeOH, acetone, and CH2Cl2, which yielded eight fractions. Subfraction 2.1.7.4 contained compound 2, while compounds 3 and 4 were found in subfraction 2.1.7.5. Compound 2 (27-O-methylasporyzin C) was isolated via RP18-HPLC (Nucleodur 100-S 250 mm × 4.6 mm) with MeOH−H2O (82:18), 1.2 mL min−1, as a white powder, tR 12 min (2.0 mg). Subfraction 2.1.7.5 was further purified via RP18-HPLC (Nucleodur 100-S 250 mm × 4.6 mm) with MeOH−H2O (87.5:12.5), 1.3 mL min−1. Compound 3 (JBIR-03) was isolated as a yellowishwhite powder [tR 23 min] (30.0 mg) and compound 4 (emindole SB) as a white powder [tR 21 min] (7.2 mg). Emindole SB beta-mannoside (1): white, amorphous compound (6.5 mg; 0.65 mg L−1); [α]25D +33.1 (c 0.36, MeOH); UV (MeOH) λmax (log ε) 229 (4.27), 281 (3.67) nm; IR (ATR) νmax 3347, 2924, 1651, 1454, 1375, 1303, 1254, 1072, 1025, 740 cm−1; 1H and 13C NMR data (see Table 1); ESIMS m/z 568 [M + H]+, 586 [M + NH 4 ] + ; HRESIMS m/z 590.3440 [M + Na] + (calcd for C34H49NO6Na, 590.3452). 27-O-Methylasporyzin C (2): white, amorphous compound (2.0 mg; 0.2 mg L−1); [α]25D −27.6 (c 0.17, MeOH); UV (MeOH) λmax (log ε) 229 (5.31), 280 (4.76) nm; IR (ATR) νmax 3361, 2924, 1642, 1585, 1376, 1202, 1072, 1376, 1202, 668, 631 cm−1; 1H and 13C NMR data (see Table 1); ESIMS m/z 436 [M + H]+; HRESIMS m/z 458.3043 [M + Na]+ (calcd for C29H41NO2Na, m/z 458.3030). JBIR-03 (3): white, amorphous compound (30.0 mg, 3.0 mg L−1); [α]25D −18.1 (c 1.10, CH3CN) [lit. [α]24.5D +46.2 (c 0.05, MeOH)14]; 1 H and 13C NMR data (see Table S3); ESIMS m/z 404 [M + H]+. Emindole SB (4): white, amorphous compound (7.2 mg, 0.72 mg L−1); [α]25D −24.2 (c 0.12, acetone) [lit. [α]25D −19 (c 0.2, CHCl3)10]; 1H and 13C NMR data (see Table S3); ESIMS m/z 406 [M + H]+. Acid Hydrolysis and Chiral-Phase HPLC. To 2 mg of compound 1 was added 2 mL of 2 M HCl, and the resulting solution was heated at 80 °C for 5 h under reflux. The residual solvent was removed under reduced pressure, and the residue was dissolved in 1 mL of EtOH. This solution was used for chiral-phase HPLC analysis using the Merck HPLC system with the ELSD detector under the following conditions: column, Diacel CHIRALPAK IA column (250 mm × 4.6 mm, 5 μm); mobile phase, hexane−EtOH (70:30); flow, 0.5 mL min−1. Using these conditions the α-D-mannose had a retention time of 11.2 min, β-D-mannose of 17.0 min, α-L-mannose of 13.1 min, and β-L-mannose of 22.4 min, respectively (Supporting Information, Figures 10.1−10.4). Bioassays. Radioligand Binding Studies at CB 1 and CB2 Receptors. Competition binding assays were performed versus the cannabinoid receptor agonist radioligand [3H](−)-cis-3-[2-hydroxy-4(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl)cyclohexanol (CP55,940) as described before.16 Stock solutions of test compounds were prepared in DMSO. The final DMSO concentration in the assay was 2.5%. Data were obtained from three independent experiments, performed in duplicate. cAMP Accumulation Assays. The measurement of intracellular cAMP concentration was performed as described before.19 Inhibition of adenylate cyclase activity was determined in CHO cells stably expressing the CB1 or CB2 receptor subtype using a competition binding assay for cAMP quantification. Data were obtained from three independent experiments, performed in duplicate.
preference for the CB2 receptor subtype. Compound 1 was examined for cytotoxic activity toward an L6 rat skeletal muscle cell line. In comparison to the cytotoxic reference compound podophyllotoxin, with an IC50 of 0.017 μM, compound 1 showed no cytotoxic activity (IC50 58 μM).17,18 In addition we identified 2 and 3 as the first ligands of fungal origin for the orphan receptor GPR18. Selective GPR18 antagonists are urgently needed as pharmacological tools to further elucidate the physiological role of this poorly characterized receptor. So far, no selective antagonists for the GPR18 have been described.
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EXPERIMENTAL SECTION
General Experimental Procedures. Optical rotations were measured with a Jasco DIP 140 polarimeter. UV and IR spectra were obtained employing Perkin-Elmer Lambda 40 and Perkin-Elmer Spectrum BX instruments, respectively. 1H and 13C NMR spectra were recorded on a Bruker Avance 300 DPX spectrometer. NMR spectra were referenced to residual solvent signals of acetone and methanol at δH/C 2.04/29.8 and 3.35/49.0, respectively. LRESIMS measurements were performed employing an API 2000 Triple Quadrupole LC/MS/ MS (Applied Biosystems/MDS Sciex) with ESI source. HRESIMS were recorded on a Bruker Daltonik micrOTOF-Q time-of-flight mass spectrometer with ESI source. VLC grade (40−63 μm) Si gel was used for vacuum liquid chromatography. All organic solvents were distilled prior to use. HPLC was carried out using different HPLC systems: (A) Waters system, controlled by Waters Millenium software, consisting of a 600E pump, a 996 PDA detector, and a 717 Plus autosampler; (B) Merck Hitachi system, controlled by Merck Hitachi D-7000 Chromatography Data Station software version 4.0, consisting of an L6200 A intelligent pump, a D 6000 interface, and an L4500 PDA or ELSD Sedex 85 detector; (C) HP system, controlled by HP ChemStation for LC Rev.A.06.03[909] software, consisting of an L7100 Merck Hitachi pump and an HP-series 1050 detector; (D) HPLC composed of a Waters 515 pump together with a Knauer K2300 differential refractometer. Cultivation, Extraction, and Isolation. The marine-derived fungus Dichotomomyces cejpeii was isolated from a sample of the sponge Callyspongia sp. cf. C. f lammea (collected at Bear Island, Sydney, Australia). The isolation of the fungus was carried out using an indirect isolation method. Sponge samples were rinsed three times with sterile H2O. After surface sterilization with 70% EtOH for 15 s the sponge was rinsed in sterile artificial seawater (ASW). Subsequently, the sponge material was aseptically cut into small pieces and placed on agar plates containing isolation medium: agar 15 g L−1, ASW 800 mL L−1, glucose 1 g L−1, peptone from soymeal 0.5 g L−1, yeast extract 0.1 g L−1, benzylpenicillin 250 mg L−1, and streptomycin sulfate 250 mg L−1. The fungus growing out of the spongeal tissue was separated on biomalt medium (biomalt 20 g L−1, agar 10 g L−1, ASW 800 mL L−1) until the culture was pure. The isolated fungus was identified by P. Massart and C. Decock, BCCM/ MUCL, Catholic University of Louvain, Belgium. A specimen is deposited at the Institute for Pharmaceutical Biology, University of Bonn, isolation number 293K09, strain number 225. The fungus was cultivated for 30 days on 10 L of malt, peptone, yeast solid agar medium (malt extract 20 g L−1, peptone 2.5 g L−1, yeast extract 2.5 g L−1, and agar 15 g L−1) supplemented with demineralized H2O in 40 Fernbach flasks at room temperature (rt). Fungal biomass and media were homogenized using an UltraTurrax apparatus and extracted with 5 L of EtOAc to yield 4.1 g of extract. This material was fractionated by NP vacuum liquid chromatography (VLC) using a stepwise gradient solvent system of increasing polarity starting with 100% CH2Cl2 to 100% EtOAc and to 100% MeOH, which yielded 10 fractions. Compound 1 (emindole SB beta-mannoside) was isolated from VLC fraction 7, while compounds 2, 3, and 4 were found in VLC fraction 2. Fraction 7 was separated via RP C18-VLC using stepwise elution with MeOH−H2O (70:30) to 100% MeOH, which yielded seven 676
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β-Arrestin-Recruitment Assays. Recruitment of β-arrestin to the respective GPCR was detected by using the β-galactosidase enzyme fragment complementation technology (β-arrestin PathHunter assay, DiscoverX) as previously described.16 CHO cells stably expressing the respective receptor were seeded in a volume of 90 μL into a 96-well plate and were incubated at a density of 20 000 cells/well in assay medium (Opti-MEM, 2% FCS, 100 U mL−1 penicillin, 100 μg mL−1 streptomycin, 800 μg mL−1 Geneticin, and 300 μg mL−1 hygromycin) for 24 h at 37 °C. Test compounds were diluted in PBS buffer containing 10% DMSO and 0.1% BSA, and 10 μL was added to 90 μL of the cell culture, followed by an incubation for 90 min at 37 °C. For determination of β-arrestin recruitment to GPR18, a commercial detection reagent was used, according to the suppliers protocol (DiscoverX). The detection reagent for GPR55 was prepared by mixing the chemiluminescent substrate Galacton-Star (2 mM) with the luminescence enhancer Emerald-II and a lysis buffer (10 mM TRIS, 1 mM EDTA, 100 mM NaCl, 5 mM MgCl2, 1% Triton-X; pH 8) in a ratio of 1:5:19. After the addition of 50 μL/well of detection reagent to the cells, the plate was incubated for a further 60 min at rt. Finally luminescence was determined in a luminometer (TopCount NXT, Packard/Perkin-Elmer). Screening for antagonism of test compounds was performed in the presence of 10 μM Δ9-THC (GPR18) or 1 μM LPI (GPR55), respectively. IC50 values of compounds were determined in the presence of a constant concentration of 7.5 μM Δ9-THC (GPR18) or 1 μM LPI (GPR55), respectively. Data were obtained from three independent experiments, performed in duplicate. In Vitro Cytotoxicity with L6 Cells. Assays were performed in 96well microtiter plates, each well containing 100 μL of RPMI 1640 medium supplemented with 1% L-glutamine (200 mM) and 10% fetal bovine serum and 4000 L6 cells (a primary cell line derived from rat skeletal myoblasts).20 Serial drug dilutions of 11 3-fold dilution steps covering a range from 100 to 0.002 μg mL−1 were prepared. After 70 h of incubation the plates were inspected under an inverted microscope to ensure growth of the controls and sterile conditions. Alamar Blue (10 μL) was then added to each well, and the plates were incubated for another 2 h. Then the plates were read with a Spectramax Gemini XS microplate fluorometer (Molecular Devices, Sunnyvale, CA, USA) using an excitation wavelength of 536 nm and an emission wavelength of 588 nm. The IC50 values were calculated by linear regression from the sigmoidal dose−inhibition curves using SoftmaxPro software (Molecular Devices). Podophyllotoxin (Sigma P4405) was used as the control.18
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Note
DEDICATION Dedicated to Prof. Dr. Otto Sticher of ETH-Zurich, Zurich, Switzerland, for his pioneering work in pharmacognosy and phytochemistry.
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REFERENCES
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ASSOCIATED CONTENT
S Supporting Information *
Compound characterization, 1H and 13C NMR spectra, and purity data for compounds 1−4 and chiral HPLC chromatograms. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Authors
*Tel: +49228732301. Fax: +49228732567. E-mail: christa.
[email protected]. *Tel: +49228733747. Fax: +49228733250. E-mail: g.koenig@ uni-bonn.de. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS Financial support came from the Deutsche Forschungsgemeinschaft (FOR 854) and from the NRW International Research Graduate School Biotech-Pharma. 677
dx.doi.org/10.1021/np400850g | J. Nat. Prod. 2014, 77, 673−677
