Penicillic Acid Congener,a New Inhibitor of BMP-Induced Alkaline Phosphatase Activity in Myoblasts,Produced by the Fungus Penicillium sp. BF-0343

Abstract

During our screening for microbial regulators of bone metabolism, a new compound, 6-ethoxy-5,6-dihydropenillic acid (1), was isolated together with a known and structurally related penicillic acid (2) from the culture broth of the soil-derived fungus Penicillium sp. BF-0343. The structure of 1 was elucidated by various spectroscopic data including nuclear magnetic resonance experiments. Compounds 1 and 2 dose-dependently inhibited bone morphogenetic protein–induced alkaline phosphatase activity in myoblasts with half-maximal inhibitory concentration values of 19.8 and 2.1 μM, respectively.

Our group has been interested in the discovery of new compounds from microorganisms. ^{1 -6} One of our research interests is to search for microbial regulators of bone metabolic disorders. For example, fibrodysplasia ossificans progressiva (FOP) is an orphan disease characterized by progressive and widespread heterotopic ossification in soft tissues, such as muscle, tendons, and ligaments. ^7,8 Surgical resection of heterotopic bone is prohibited in FOP patients because injury to soft tissues induces severe heterotopic ossification; thus, drug therapies are required. To date, several compounds have been reported as potential therapeutic agents for FOP patients; however, none of them have yet been approved. ^{9 -13} A mutation in the ACVR1/ALK2 gene, a bone morphogenetic protein (BMP) type I receptor, has been identified as causative of FOP. ¹⁴ This mutation results in the amino acid substitution of Arg206 to His (R206H) within ACVR1/ALK2 and is found in over 90% of FOP patients. Previous studies have clearly shown that this mutation is a gain-of-function mutation that activates intracellular BMP signaling to induce heterotopic ossification. ¹⁵ Therefore, inhibitors of BMP signaling might offer therapeutic benefits for FOP patients. Based on this background, we screened microbial culture broths for inhibitors of BMP-induced alkaline phosphatase (ALP) activity using constitutively active ALK2 (R206H)-expressing C2C12 myoblasts (abbreviated as C2C12 (R206H)) and discovered several microbial inhibitors. ^{16 -18} By our continuous screening efforts, a new compound, 6-ethoxy-5,6-dihydropenicillic acid (1), was isolated together with a known and structurally related penicillic acid (2) from a culture of the fungal strain Penicillium sp. BF-0343 (Figure 1). In the present paper, the fermentation, isolation, structural elucidation, and biological activity of 1 are described.

Figure 1

Structures of 1 and 2.

Results and Discussion

The fungal strain BF-0343 was originally isolated from a soil sample collected at Fuji Cemetery, Shizuoka, Japan. In a BLAST search from the International Nucleotide Sequence Databases, the 28S rDNA-D1/D2 and internal transcribed spacer (ITS)-5.8S rDNA of the fungus BF-0343 had high similarity with those of Penicillium species such as Penicillium brasilianum, P. onobense, and P. skrjabinii (98.4% to 99.8%). Thus, this strain was considered to belong to the genus Penicillium.

Compound 1 was obtained as a pale-yellow oil. In the ultraviolet (UV) spectrum, 1 showed an absorption maximum at 224 nm. Infrared (IR) absorption at 3425 and 1645 cm⁻¹ suggested the presence of hydroxyl and carbonyl groups in the structure. The molecular formula of 1 was assigned as C₁₀H₁₆O₅ on the basis of high-resolution electron ionization-mass spectrometry (EI-MS) measurements ([m/z 216.0990 (M)⁺ m/z Δ −0.7 mmu]).

The ¹H nuclear magnetic resonance (NMR) spectrum (Table 1) in CHCl₃-d displayed 16 proton signals, 2 of which were assigned to a hydroxyl proton (1 H, δ 7.01) and methoxy protons (3 H, δ 3.91). Its ¹³C NMR spectrum revealed 10 carbon resonances, corresponding to 3 sp ³ methyl carbons, 2 sp ³ methylene carbons, 1 sp ³ methine carbon, 1 sp ² methine carbon, 1 sp ³ quaternary carbon, 1 sp ² quaternary carbon, and 1 carbonyl carbon. Then, the partial structures I and II were established by ¹H-¹H correlation spectroscopy correlations (COSY) as shown by the bold lines in Figure 2. Furthermore, heteronuclear multiple bond correlations (HMBC) revealed the following evidences (Figure 2): (1) The cross peaks from H-7 (δ 3.50-3.65 and 3.82) to C-9 (δ 105.7) and from H-9 (δ 3.50-3.65) to C-7 (δ 72.9) indicated the presence of a 1-ethoxypropane moiety including 2 partial structures. (2) The cross peaks from H-2 (δ 5.05) to C-1 (δ 170.4) and C-4 (δ 105.7), from H-8 (δ3.91) to C-3 (δ 177.9), and from OH-4 (δ 7.01) to C-3 (δ 177.9) and C-4, and the chemical shift values of C1 to C4 indicated the presence of a 3-methoxy-4-hydroxy-fruanone ring. (3) The cross peaks from H-6 (δ 0.71) and H-7 (δ 3.50-3.65 and 3.82) to C-4 (δ 105.7) indicated the linkage of 1-ethoxypropane at the C-4 position. Totally, 1 was elucidated as 6-ethoxy-5,6-dihydropenicillic acid (Figure 1). Previously, 2 groups reported 5,6-dihydro-6-hydroxy penicillic acid and its analogs. ^19,20 In the present study, we found a new analog in this family. The structure of 2 was similarly elucidated by MS and NMR experiments and identified as penicillic acid by comparison with previous literature. ²¹ We suspected that 1 might be generated through an addition reaction of ethanol into 2, whereas such an adduct was not observed when we treated 2 with ethanol under the experimental conditions.

Figure 2

Key ¹H-¹H COSY and HMBC correlations of 1. COSY, correlation spectroscopy; HMBC, heteronuclear multiple bond correlation.

Table 1

¹H and ¹³C Nuclear Magnetic Resonance Chemical Shifts of 1.

Position	δ C	δ H (mult., J in Hz)
1	170.4
2	89.7	5.06 (s)
3	177.8
4	105.7
4-OH		7.01 (s)
5	36.5	2.50 (m)
6	11.2	0.71 (d, 6.8)
7	72.9	3.50-3.65 (m) a , 3.82 (dd, 11.2, 9.2)
8	59.5	3.91 (s)
9	67.3	3.50-3.65 (m) a
10	14.9	1.23 (t, 6.8)

^aOverlapping signals.

According to the published method, ¹⁵ the effects of 1 and 2 on BMP-induced ALP activity, a typical marker of osteoblastic differentiation, were evaluated. Their cell cytotoxicity was also evaluated by MTT. As summarized in Table 2, compounds 1 and 2 dose-dependently inhibited BMP-induced ALP activity with half-maximal inhibitory concentration values of 19.8 and 2.1 µM, respectively. Meanwhile, the cytotoxic effects of these compounds on C2C12 cells (R206H) were much less.

Table 2

Effects of 1 and 2 on ALP Activity and Cell Cytotoxicity.

Compound	IC50 (µM)
	ALP	Cytotoxicity
1	19.8	558.3
2	2.1	51.8

Abbreviations: ALP, alkaline phosphatase; IC₅₀, half-maximal inhibitory concentration.

Experimental

Materials

Diethanol amine, sodium dodecyl sulfate (SDS), HCl, MgCl₂, NaCl, and NaOH were purchased from Wako Pure Chemical Industries (Osaka, Japan). Acetic acid, acetonitrile, Dulbecco’s modified Eagle’s medium, ethyl acetate, methanol, and N,N-dimethylformamide were obtained from Nacalai Tesque (Kyoto, Japan). NaSO₄ was from Kanto Chemical (Tokyo, Japan). Potato dextrose broth was purchased from Becton Dickinson (Sparks, MD, USA). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium and p-nitrophenyl phosphate were obtained from Sigma (St Louis, MO, USA). Penicillin/streptomycin was from Invitrogen (Carlsbad, CA, USA). Fetal bovine serum was purchased from Hyclone (Waltham, MA, USA). Recombinant human BMP4 (rhBMP4) was obtained from R&D Systems (Mountain View, CA, USA). Ethanol was from Japan Alcohol Sales (Tokyo, Japan). CHCl₃-d was purchased from Acros Organics (Geel, Antwerpen, Belgium). PEGASIL ODS SP100 was obtained from Senshu Scientific (Tokyo, Japan) for analysis using high-performance liquid chromatography (HPLC). Ninety-six-well plastic plates were from Corning (Corning, NY, USA).

General Experimental Procedures

The genus of the fungus BF-0343 was identified based on a genetic analysis of an rDNA ITS by the identification services of TechnoSuruga Laboratory Co., Ltd., Shizuoka, Japan. This strain was used to produce 1 and 2. EI-MS spectrometry was conducted on a JEOL JMS-AX505HA spectrometer (JEOL, Tokyo, Japan). UV spectra were measured with a U-2800 spectrophotometer (Hitachi, Tokyo, Japan). IR spectra were measured with a FT/IR-460 plus spectrometer (JASCO, Tokyo, Japan). The ¹³C (100 MHz) and ¹H (400 MHz) spectra of the compounds were taken on a 400-MR spectrometer (Agilent Technologies, Santa Clara, CA, USA) in CHCl₃-d, and the solvent peak was used as the internal standard at 7.26 ppm for ¹H NMR and 77.0 ppm for ¹³C NMR.

Fermentation

The fungus BF-0343 was grown on potato dextrose agar medium (potato dextrose broth 2.4% and agar 1.5%, pH 6.0), and then used to inoculate a 50-mL test tube containing 15 mL seed medium (potato dextrose broth 2.4% and 0.1% agar, pH 6.0). The test tube was shaken on a reciprocal shaker at 27 °C for 3 days. A 1-mL portion of the seed culture was then inoculated into a 500-mL Erlenmeyer flask containing 100 mL of the production medium (potato dextrose broth 2.4%, pH 6.0). The fermentation was performed on a rotary shaker at 27 °C for 7 days.

Isolation

The whole broth (100 mL) was fermented for 7 days and then treated with an equal volume of ethanol. After the extracts were filtrated and concentrated to remove ethanol, the aqueous resultant was adjusted to pH 6.0 with 2N sodium hydroxide and extracted with an equal volume of ethylacetate. The organic layer collected was sufficiently dehydrated with sodium sulfate and concentrated in vacuo to give a brown material (61.1 mg). This material was dissolved in a small amount of methanol and finally purified with preparative HPLC under the following conditions: column, PEGASIL ODS SP100 (internal diameter 20 × 250 mm); mobile phase, 20% CH₃CN; detection, UV at 210 nm; flow rate, 6 mL/mL. Under these conditions, 1 and 2 were eluted as peaks with retention times of 15 and 22 minutes, respectively. The fractions pooled were concentrated in vacuo to dryness to yield pure 1 (7.5 mg) and 2 (26.2 mg).

Cell Culture

The C₂C₁₂ (R206H) myoblast cell line was cultured in Dulbecco’s modified Eagle’s medium supplemented with 15% fetal bovine serum, 100 units/mL penicillin, and 100 µg/mL streptomycin (hereafter referred to as medium A) at 37 °C in 5% CO₂. Cells were subcultured once every 3 days.

Assay for ALP in BMP-Treated C2C12 (R206H) Cells

ALP activity, a typical marker of osteoblastic differentiation, was measured as previously described. ¹⁵ In brief, C2C12 (R206H) cells (7.5 × 10³ cells per well) in a 96-well plastic plate were cultured at 37 °C in 5% CO₂. Following overnight recovery, the culture media were replaced with 100 µL fresh medium A containing rhBMP4 (10 ng/mL) and a sample (1 µL in MeOH solution). After 48-hour incubation, the cells were fixed with acetone-ethanol (1:1) solution and then incubated with 100 µL substrate solution (100 mM diethanolamine, 0.5 mM MgCl₂, and 1.0 mg/mL p-nitrophenyl phosphate) at room temperature. The reaction was terminated by adding 50 µL of 3 M NaOH, and the absorbance of 405 nm was measured with a Power Wave x340 (BIO-TEK Instruments, Highland Park, IL, USA).

Cytotoxicity

The cytotoxicity of a compound to C2C12 (R206H) cells was evaluated by the MTT assay. In brief, C2C12 (R206H) cells (7.5 × 10³ cells per well) were cultured in 96-well plates in the absence or presence of a compound for 48 hours at 37 °C in 5% CO₂. After incubation, the cells received 10 µL MTT solution (5.5 mg/mL in phosphate-buffered saline) and were then incubated at 37 °C for 3 hours. A 90-µL aliquot of lysis solution (40% N, N-dimethylformamide, 2.0% CH₃COOH, 20% SDS, and 0.03 M HCl) was added to each well, and the plates were incubated for 2 hours. The absorbance at 570 nm of each well was read with a Power Wave x340.

Compound 1: Appearance, pale yellow oil; UV (MeOH) λmax (ε), 224 nm (7614); IR (KBr) νmax (cm⁻¹), 3425, 2923, 1747, 1645, 1429, 1070; HR-EI-MS m/z 216.0990 (M⁺, C₁₀H₁₆O₅ calculated 216.0998); [α]_D ²⁴ (c = 0.2, CHCl₃), −0.8; ¹H and ¹³C NMR, Table 1. Please see Supplemental File about the NMR and MS spectra.

Supplemental Material

Supplementary Material 1 - Supplemental material for Penicillic Acid Congener, a New Inhibitor of BMP-Induced Alkaline Phosphatase Activity in Myoblasts, Produced by the Fungus Penicillium sp. BF-0343

Supplemental material, Supplementary Material 1, for Penicillic Acid Congener, a New Inhibitor of BMP-Induced Alkaline Phosphatase Activity in Myoblasts, Produced by the Fungus Penicillium sp. BF-0343 by Nobuhiro Koyama, Yasuhiro Otoguro, Satoshi Ohte, Takenobu Katagiri and Hiroshi Tomoda in Natural Product Communications