Nonactic Acid Derivatives Isolated From Streptomyces werraensis IFM12104 in a Screening Program for BMI1 Promoter Inhibitory Activity

Abstract

Upon screening for compounds with B cell-specific Moloney murine leukemia virus region 1 (BMI1) promoter inhibitory activity, 3 nonactic acid derivatives (1-3) were isolated from Streptomyces werraensis IFM12104. Although spectral data of compound 1 were identical with those reported in literature for 4α-(3, 5-dihydroxy-heptyl)-3α-methyl-2-oxetanone (4), compound 1 was revealed to be homononactic acid by X-ray analysis. Compounds 2 and 3 were identified as bishomononactic acid and nonactic acid using spectral data. The 3 compounds 1 to 3 inhibited BMI1 promoter activities with IC₅₀ values of 240, 160, and 180 μM, respectively.

Polycomb repressive complex 1 (PRC1) is an epigenetic regulator of gene expression. ¹ One of the central component proteins of PRC1 is B cell-specific Moloney murine leukemia virus region 1 (BMI1). Overexpression of BMI1 is closely linked to the development of specific cancers, ² and inhibitors of BMI1 may potentially be speculated as effective cancer therapeutic candidates.

In our previous study, a reporter gene assay system that evaluates the promoter activity of BMI1 was constructed. ³ In mammalian cells, transcription factor c-Myc binds to the BMI1 promoter and regulates expression of the BMI gene downstream the BMI1 promoter. ⁴ In this study, a human embryonic kidney 293T cell line, stably transfected with a reporter plasmid carrying a luciferase-encoding gene inserted downstream the BMI1 promoter instead of the BMI1 gene, was used for the assay system. The BMI1 promoter activity was evaluated by measuring the luciferase expression levels of this cell line (BMI1/293T) (Figure 1). Using this assay system, 7 compounds including somalin, maslinic acid, digitoxigenin, and wallichoside were identified as BMI1 promoter inhibitors from 2 plant extracts. ³ Recently, 3 compounds, elaiophylin, 2-methylelaiophylin, and nocardamin, were isolated from Streptomyces sp. IFM-11958 that inhibited BMI1 promoter activity. ⁵ However, existence of only one report on BMI1 promoter inhibitors from actinomycetes encouraged us to further investigate them.

Figure 1

Constructed BMI1 promoter assay system.

Results and Discussion

Upon screening the actinomycetes extracts collected in Japan by the BMI1 promoter assay system, the extract of Streptomyces werraensis IFM12104 was identified as a positive hit possessing BMI1 promoter inhibitory activity. This strain was cultured in Waksman liquid medium ⁶ (12 L) at 28°C for 5 days. After centrifugation of the culture extract, the mycelium cake was extracted with methanol (MeOH). Fractionation of the MeOH extract was guided by BMI1 promoter inhibitory activity test. The MeOH extract was partitioned with ethyl acetate (EtOAc)/H₂O to give a layer of EtOAc, which was fractionated by silica gel and Reverse Phase (RP)- High Performance Liquid Chromatography (HPLC) to provide compounds 1 to 3 (Figure 2).

Figure 2

Compounds 1 to 3 isolated from Streptomyces werraensis IFM12104 and compound 4.

Compound 1 was revealed to have the molecular formula C₁₁H₂₀O₄ on the basis of ¹H and ¹³C Nuclear Magnetic Resonance (NMR) data as well as from Mass spectrometry (MS) results. Detailed analysis further suggested that the ¹H and ¹³C NMR and MS data of compound 1 were identical with those reported in the literature ⁷ for the compound 4α-(3, 5-dihydroxy-heptyl)-3α-methyl-2-oxetanone (compound 4), which was isolated from Streptomyces sp. T1B1 (Figure 2). The structure of compound 4 has β-lactone moiety with side chain containing 1, 3-diol moiety. The stereochemistry of 1, 3-diol moiety remains undetermined thus far. Therefore, to determine the stereochemistry, X-ray crystallographic analysis was performed for compound 1 obtained in this study (CCDC 1921357, Figure 3, and Supporting information, Tables S1-S13). Compound 1 was revealed not to possess β-lactone structure and its structure was identical with that of homononactic acid, previously isolated from Streptomyces griseus LKS-1 ⁸ (Table 1). Upon comparison of spectral data with those described in the literature, compounds 2 and 3 isolated in this study were identified as bishomononactic acid and nonactic acid, respectively. ⁹ Interestingly, compound 1 was isolated in its racemic form because optical rotation showed the minimum value ( ${\displaystyle [\alpha {]}_{\mathrm{D}}^{21}}$ + 0.93) and the space group of crystal of compound 1 was determined to be C2/c (#15), which is known to be one of the space groups representing racemic crystals, ¹⁰ whereas compounds 2 ( ${\displaystyle [\alpha {]}_{\mathrm{D}}^{21}}$ + 15.0) and 3 ( ${\displaystyle [\alpha {]}_{\mathrm{D}}^{22}}$ +11.0) were isolated as optical active forms.

Figure 3

The X-ray structure of compound 1.

Table 1

Comparison of ¹H and ¹³C NMR Data Between Compounds 1 and 4.

Position	δ H (J in Hz)		δ C
	Homononactic acid (1)	4α-(3, 5-Dihydroxy-heptyl)- 3α-methyl-2-oxetanone (4) a	Homononactic acid (1)	4α-(3, 5-Dihydroxy-heptyl)- 3α-methyl-2-oxetanone (4) a
1			178.0	177.8
2	2.52 (quin, 7.3)	2.54 (m)	45.4	45.3
3	4.03 (q, 7.3)	4.02 (q, 7.6)	81.0	81.1
4	2.05 (m)1.70 (m)	2.04 (m) 1.67 (m)	28.9	29.1
5	2.03 (m)1.67 (m)	2.02 (m) 1.67 (m)	30.5	30.5
6	4.23 (m)	4.23 (m)	77.1	77.0
7	1.74 (m)	1.73 (m)	40.7	40.6
8	3.80 (m)	3.79 (m)	70.2	70.4
9	1.54 (brm, 2 H)	1.59 (m, 2 H)	29.7	29.8
10	0.95 (t, 7.3, 3 H)	0.95 (t, 9.2, 3 H)	10.0	10.1
11	1.18 (d,7.3, 3 H)	1.17 (d, 8.6, 3 H)	13.6	13.7

Measured in CDCl₃, 600 MHz (¹H), 150 MHz (¹³C).

δ in ppm, J in Hz.

a

The chemical shifts of 4α-(3,5-dihydroxy-heptyl)-3α-methyl-2-oxetanone were taken from Ref. 7.

The compounds 1 to 3 inhibited BMI1 promoter activity with IC₅₀ values of 240, 160, and 180 µM, respectively (Figure 4). The difference in the strength of BMI1 promoter inhibitory activity between compounds 1 and 3 may depend on the carbon chain length at position 9 and whether the terminal carbon is linear or branched.

Figure 4

BMI1 promoter activity and cell viability of compounds 1 to 3. DMSO was used as a negative control (cont.) and 100 µM of c-Myc Inhibitor II 10074-G519 was used as a positive control (II). DMSO, dimethyl sulfoxide.

In this study, we described the isolation of 3 nonactic acid derivatives having BMI1 promoter inhibitory activity from S. werraensis IFM12104. In addition, the X-ray analysis of compound 1 suggested that the reconsideration of its assignment for the β-lactone containing structure (4) described in the literature ⁷ may be necessary.

Experimental

General Experimental Procedures

The following instruments were used in this study: a P-2200 polarimeter (JASCO, Tokyo) for optical rotations; an ECZ-600 spectrometer (JEOL, Tokyo) for NMR spectroscopy (solvent chemical shifts were used as the internal standard); a PU 980 (JASCO) pump and UV 970 (JASCO) for HPLC; a liquid chromatography-mass spectrometry (LC-MS) system including LCMS 2020 (Shimadzu, Kyoto) for Electrospray ionization (ESI) MS; and a MicroMax-007HF (Rigaku, Tokyo) for X-ray crystallographic analysis. The chromatographic data were collected and processed using LabSolution software (Shimadzu). The following adsorbents were used for purification: Silica gel 60 F₂₅₄ (0.25 mm, Merck, Darmstadt) and Silica gel 60 RP-8 F_254S (0.25 mm, Merck) for analytical TLC; Silica gel 60 N (Kanto Chemical Co., Inc., Tokyo) for column chromatography; and COSMOSIL 5C₁₈-AR-II (10.0 × 250 mm, Nacalai Tesque, Kyoto) for preparative HPLC.

Identification of Bacterial Strain

Streptomyces werraensis IFM12104 was collected from soil of Sakuragi cemetery, Chiba city in Japan. The identification of S. werraensis IFM12104 was performed by Dr Takashi Yaguchi at the Medical Mycology Research Center, Chiba University, where a voucher specimen was deposited with the code IFM12104. This strain was identified on the basis of the 16S rRNA gene sequencing.

Fermentation of Streptomyces werraensis IFM12104

Streptomyces werraensis IFM12104 was cultivated on Waksman agar medium consisting of 2.0% glucose, 0.5% meat extract, 0.5% peptone, 0.5% NaCl, 0.3% dried yeast, 0.3% CaCO₃, and 1.5% agar. A total of 3 colony on agar was transferred to 3 test tubes containing 5 mL of Waksman liquid medium, cultured with shaking (160 rpm) at 28°C for 7 days. The resulting cultures (5 mL each) were next added to 3 500-mL Sakaguchi flasks containing 100 mL of Waksman liquid medium, each, and grown at 28°C for 7 days with shaking at 200 rpm. Each of the resulting 100 mL cultures was then added to 12 Turnip-shaped flasks (capacity 3 L) having 1 L Waksman liquid medium and grown with shaking (200 rpm) at 28°C for 7 days. This culture broth (12 L) was subsequently centrifuged at 6000 rpm for 10 minutes to obtain the supernatant and mycelium cake. The mycelium cake was extracted thrice with MeOH (0.8 L) to obtain the mycelium MeOH extract (23.9 g).

Extraction and Isolation

The mycelium MeOH extract (23.9 g) was partitioned with EtOAc (0.3 L × 3) and water. The EtOAc layer showed inhibitory of BMI1 promoter activity (Supporting information, Figure S1). The EtOAc layer (1.1 g) was subjected to silica gel column chromatography (ϕ30 × 210 mm, CHCl₃–MeOH system) to give fractions 1A–1I. A part of the fraction 1F (CHCl₃: MeOH = 12:1, 50 mg of 234.6 mg) was subjected to reverse-phase HPLC (COSMOSIL 5C₁₈-AR-II [ϕ10.0 × 250 mm]; eluent: 60% MeOH containing 0.1% HCOOH; flow rate: 3.0 mL/min; UV detection: 360 nm) to give compounds 1 (23.3 mg, t _R 9.0 minutes) and 2 (6.0 mg, t _R 14.0 minutes). A part of the fraction 1D (CHCl₃: MeOH = 8:1, 75 mg of 342.2 mg) was subjected to reverse-phase HPLC (COSMOSIL 5C₁₈-AR-II [ϕ10.0 × 250 mm]; eluent: 60% MeOH containing 0.1% HCOOH; flow rate: 2.0 mL/min; UV detection: 254 nm) to give compounds 3 (21.5 mg, t _R 10.0 minutes) and 1 (24.0 mg, t _R 13.0 minutes).

Homononactic Acid (1)

Racemic crystal. ESIMS m/z: 217 [M + H]+. ${\displaystyle [\alpha {]}_{\mathrm{D}}^{21}}$ + 0.93 (c 0.15, CHCl3). ¹H and ¹³C NMR data are provided in Table 1.

Bishomononactic Acid (2)

White solid. ESIMS m/z: 231 [M + H]+. ${\displaystyle [\alpha {]}_{\mathrm{D}}^{21}}$ +15.0 (c 0.08, CHCl3). ¹H and ¹³C NMR data were identical with those in the literature. ⁹

Nonactic Acid (3)

White solid. ESIMS m/z: 203 [M + H]+. ${\displaystyle [\alpha {]}_{\mathrm{D}}^{22}}$ + 11.0 (c 0.15, CHCl3). ¹H and ¹³C NMR data were identical with those in the literature. ⁹

Luciferase Assay (BMI1 Promoter Activity Assay)

BMI1/293T cells (3 × 10⁴ cells/100 µL/well) were seeded in a 96-well flat bottom white microplate and precultured in a CO₂ incubator at 37°C. After incubation for 24 hours, the old medium was removed from each well, followed by addition of 200 µL of sample containing Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS) adjusted to an appropriate concentration to each well. The cells were cultured for 24 hours. After the removal of the old medium, the cells were washed once with 200 µL of phosphate buffered saline (PBS), and 30-µL Cell Culture Lysis Reagent 1 × solution (Promega, Wisconsin) was added to each well. After shaking for 30 minutes, 100 µL of Luciferase Assay Substrate (Promega) dissolved in Luciferase Assay Buffer (Promega) was added to each well to measure chemiluminescence by a Luminoscan Ascent (Thermo Fisher Scientific, Massachusetts). Data are presented as mean ± SD of n = 3 independent experiments. Dimethyl sulfoxide (DMSO) and 100 µM of c-Myc Inhibitor II 10074-G5 ¹¹ (Merck) were used as the negative and positive controls, respectively.

Cell Viability Assay

Cell viability was measured by the fluorometric microculture cytotoxicity assay method. ¹² BMI1/293T (3 × 10⁴ cells/well) were seeded onto 96-well black microplate and precultured in a CO₂ incubator at 37°C. After incubation for 24 hours, the medium was removed and 200 µL of sample containing DMEM with 10% FBS adjusted to an appropriate concentration was added per well. The cells were cultured for 24 hours. After removing the medium, the cells were washed once with 200 µL of PBS, and 200 µL of the fluorescein diacetate (Wako, Osaka) solution (3.5 µg/mL) was added to each well. After incubation for 1 hour, fluorescein fluorescence was measured (excitation: 485 nm, emission: 538 nm). Data are presented as mean ± SD of n = 3 independent experiments. DMSO was used as a negative control in this experiment.

Preparation of Single Crystals of Compound 1

Compound 1 (6 mg) was dissolved in 1.0 mL of diisopropyl ether (Wako) and heated at 50°C for 30 seconds. This solution was then cooled at 4°C for 4 days. Unit cell constants and intensities of reflections were measured on a Rigaku R-AXIS RAPID diffractometer (θ/2θ- scanning) using CuKα-radiation (λ = 1.54187 Å). The structures were solved by using CrystalStructure crystallographic software, and refined using SHELXL-97 programs.

Crystallographic Data

CCDC1921357 contains the supplementary crystallographic data of compound 1. This data is freely available at the Cambridge Crystallographic Data Centre via https://www.ccdc.cam.ac.uk/structures/.

Supplemental Material

Supporting Information - Supplemental material for Nonactic Acid Derivatives Isolated From Streptomyces werraensis IFM12104 in a Screening Program for BMI1 Promoter Inhibitory Activity

Supplemental material, Supporting Information for Nonactic Acid Derivatives Isolated From Streptomyces werraensis IFM12104 in a Screening Program for BMI1 Promoter Inhibitory Activity by Yusuke Yokoyama, Midori A. Arai, Yasumasa Hara, and Masami Ishibashi in Natural Product Communications