Suppression of RANKL-Induced Osteoclastogenesis by the Metabolites from the Marine Fungus Aspergillus flocculosus Isolated from a Sponge Stylissa sp.

A new α-pyrone merosesquiterpenoid possessing an angular tetracyclic carbon skeleton, ochraceopone F (1), and four known secondary metabolites, aspertetranone D (2), cycloechinulin (3), wasabidienone E (4), and mactanamide (5), were isolated from the marine fungus Aspergillus flocculosus derived from a sponge Stylissa sp. collected in Vietnam. The structures of Compounds 1–5 were elucidated by analysis of 1D and 2D NMR spectra and MS data. All the isolated compounds were evaluated for anti-proliferation activity and their suppression effects on receptor activator of nuclear factor κB ligand (RANKL)-induced osteoclast differentiation using tartate-resisant acid phosphatase (TRAP). Compounds 1–5 had no anti-proliferative effect on human cancer cell lines up to 30 μg/mL. Among these compounds, aspertetranone D (2) and wasabidienone E (4) exhibited weak osteoclast differentiation inhibitory activity at 10 μg/mL. However, mactanamide (5) showed a potent suppression effect of osteoclast differentiation without any evidence of cytotoxicity.


Introduction
Bone remodeling is the regulation to maintain the quality and mass of bone by undergoing a resorption and formation cycle repetitively [1,2]. The cells responsible for the skeleton resorption are the osteoclasts. Osteoclasts are multinucleated cells generated from their mononuclear precursor cells derived from the monocyte/macrophage lineage [3]. Osteoclasts are induced by two putative promoters, macrophage colony stimulating factor (M-CSF) and receptor activator of nuclear factor-κB ligand (RANKL). However, the excessive formation of osteoclasts causes abnormal bone remodeling, osteoporosis, rheumatoid arthritis, and periodontal disease are associated with a slight imbalance between bone formation and bone resorption [4][5][6]. Thus, efforts have been devoted to the discovery of substances that inhibit RANKL-induced osteoclast differentiation for suppressing osteoclastogenesis [7,8].
The majority of novel secondary metabolites from the ocean have structural diversities and potent bioactivities [9]. Many marine organisms, especially sponges, have been attracting significant attention from natural product chemists and biologists due to the potential of marine natural products as drugs during recent years [10]. It is known that some marine secondary metabolites from sponges may function as a chemical defense for associated microorganisms [11]. Moreover, natural products from microbial symbionts may also protect the sponge from environmental threats such as overgrowth, UV irradiation, and attack by feeder [9]. Associations between sponge and microorganisms gave rise to the idea that metabolites may be from their symbionts [12,13]. The structures of various natural products isolated from microorganisms closely resemble those identified in host animals [14,15]. As part of our continuing efforts to discover bioactive natural products from sponge-derived microorganisms, various marine sponges were collected and investigated for the isolation and identification of microorganisms. During studies on the diversity of fungi, a marine fungal strain 01NT-1.1.5, Aspergillus flocculosus derived from a sponge Stylissa sp., was isolated through experimental tools for the isolation of microorganisms. Aspergillus spp. and Penicillium spp. are well-known as the most chemically examined fungal genera [16,17]. Moreover, hundreds of structurally unique metabolites have been reported with interesting bioactivities as therapeutic candidates [18]. The fungus A. flocculosus 01NT.1.1.5 was originally isolated from the sponge Stylissa sp. collected at Nha Trang Bay, Vietnam, in February 2016. In preliminary screening, this strain showed good bioactivities such as antimicrobial and antifungal properties. The crude extract of the strain was purified further by a reversed-phase HPLC to yield one new compound, ochraceopone F (1), and four known compounds (Figures S8-S19), aspertetranone D (2) [19], cycloechinulin (3) [20], wasabidienone E (4) [21], and mactanamide (5) [22] (Figure 1). The structures of the known compounds were identified by 1D and 2D NMR analysis and comparison with literature data. All compounds were tested for anti-proliferative activity on human cancer cell lines and RANKL-induced osteoclast differentiation inhibitory effect using a TRAP assay.
All the isolated compounds (1-5) were tested against human cancer cell lines using the sulforhodamine B (SRB) assay [24] and inhibitory effects on RANKL-induced osteoclast differentiation using tartate-resistant acid phosphatase (TRAP), which is highly expressed in osteoclasts, as a primary marker. These compounds did not show any cytotoxicity against five cancer cell lines (HCT 15, NUGC-3, NCI-H23, ACHN, PC-3, and MDA-MB-231) up to 30 μg/mL. However, aspertetranone D (2), wasabidienone E (4), and mactanamide (5) showed suppressive activities on RANKL-induced differentiation of BMMs into osteoclasts as evidenced by a TRAP assay ( Figure 3B). To confirm cytotoxic effects on BMMs at their effective concentrations for the inhibition of differentiation of osteoclasts, the cell viability was measured by XTT assay ( Figure 3A). Compounds 2, 4, and 5 had no significant cytotoxic effect on BMMs at concentrations used in this study. In particular, mactanamide (5) dose-dependently suppressed RANKL-induced differentiation of BMMs into osteoclasts ( Figure 4A,B). These biological data indicate that mactanamide (5), displaying an inhibitory effect against osteoclastogenesis, might be a therapeutic drug lead for various skeletal diseases, and further research including mechanistic studies is needed. The relative configuration of 1 was established on the basis of ROESY spectrum (Figure 2 and Figure S7). to H a -15 (δ H 2.49) indicated that two methyls and the methine have different faces. The absolute configuration of ochraceopone E possessing an angular tetracyclic skeleton and having a very similar structure with 1 was determined by an X-ray diffraction study. Comparison of the optical rotation value, NMR chemical shifts, and ROESY correlations of 1 with those of ochraceopone E suggested that these compounds have the same absolute stereochemistry. This result was also supported by the fact that ochraceopones [19,23] share a biosynthetic pathway.
All the isolated compounds (1-5) were tested against human cancer cell lines using the sulforhodamine B (SRB) assay [24] and inhibitory effects on RANKL-induced osteoclast differentiation using tartate-resistant acid phosphatase (TRAP), which is highly expressed in osteoclasts, as a primary marker. These compounds did not show any cytotoxicity against five cancer cell lines (HCT 15, NUGC-3, NCI-H23, ACHN, PC-3, and MDA-MB-231) up to 30 µg/mL. However, aspertetranone D (2), wasabidienone E (4), and mactanamide (5) showed suppressive activities on RANKL-induced differentiation of BMMs into osteoclasts as evidenced by a TRAP assay ( Figure 3B). To confirm cytotoxic effects on BMMs at their effective concentrations for the inhibition of differentiation of osteoclasts, the cell viability was measured by XTT assay ( Figure 3A). Compounds 2, 4, and 5 had no significant cytotoxic effect on BMMs at concentrations used in this study. In particular, mactanamide (5) dose-dependently suppressed RANKL-induced differentiation of BMMs into osteoclasts ( Figure 4A,B). These biological data indicate that mactanamide (5), displaying an inhibitory effect against osteoclastogenesis, might be a therapeutic drug lead for various skeletal diseases, and further research including mechanistic studies is needed. The relative configuration of 1 was established on the basis of ROESY spectrum (Figure 2 and Figure S7). The ROESY correlation from H3-19 (δH 1.30) to Hb-10 (δH 2.01), Hb-10 (δH 2.01) to H3-21 (δH 1.21), H3-21 (δH 1.21) to Hb-15 (δH 2.64), and Hb-15 (δH 2.64) to H3-20 (δH 1.17) placed these protons on the same face, while the absence of a ROESY correlation between two methyls (H3-19 and H3-20) and H-7 (δH 2.47) indicated that H-7, H3-19, and H3-20 were located on different faces at the ring junctions. Additionally, ROESY correlation from H-7 (δH 2.47) to Hb-16 (δH 2.01) and Hb-16 (δH 2.01) to Ha-15 (δH 2.49) indicated that two methyls and the methine have different faces. The absolute configuration of ochraceopone E possessing an angular tetracyclic skeleton and having a very similar structure with 1 was determined by an X-ray diffraction study. Comparison of the optical rotation value, NMR chemical shifts, and ROESY correlations of 1 with those of ochraceopone E suggested that these compounds have the same absolute stereochemistry. This result was also supported by the fact that ochraceopones [19,23] share a biosynthetic pathway.
All the isolated compounds (1-5) were tested against human cancer cell lines using the sulforhodamine B (SRB) assay [24] and inhibitory effects on RANKL-induced osteoclast differentiation using tartate-resistant acid phosphatase (TRAP), which is highly expressed in osteoclasts, as a primary marker. These compounds did not show any cytotoxicity against five cancer cell lines (HCT 15, NUGC-3, NCI-H23, ACHN, PC-3, and MDA-MB-231) up to 30 μg/mL. However, aspertetranone D (2), wasabidienone E (4), and mactanamide (5) showed suppressive activities on RANKL-induced differentiation of BMMs into osteoclasts as evidenced by a TRAP assay ( Figure 3B). To confirm cytotoxic effects on BMMs at their effective concentrations for the inhibition of differentiation of osteoclasts, the cell viability was measured by XTT assay ( Figure 3A). Compounds 2, 4, and 5 had no significant cytotoxic effect on BMMs at concentrations used in this study. In particular, mactanamide (5) dose-dependently suppressed RANKL-induced differentiation of BMMs into osteoclasts ( Figure 4A,B). These biological data indicate that mactanamide (5), displaying an inhibitory effect against osteoclastogenesis, might be a therapeutic drug lead for various skeletal diseases, and further research including mechanistic studies is needed.

Fungal Material and Fermentation
The fungus A. flocculosus 01NT.1.1.5 was originally isolated from the sponge Stylissa sp. collected at Nha Trang Bay, Vietnam, in February 2016. The fungus was identified according to its gene sequences of 28S rRNA (GenBank accession number EU021616.1). A BLAST search result indicated that the sequence is similar 100% to the sequence of A. flocculosus (compared with NRRL 5224). In order to search for bioactive secondary metabolites of the strain, the fungus was grown stationary at 22 • C for 21 days in 100 Erlenmeyer flasks (500 mL), each containing 20 g of rice, 20 mg of yeast extract, 10 mg of KH 2 PO 4 , and 40 mL of natural sea water [25].  Table 1.

Osteoclastogenesis Assay
Mouse bone marrow cells were isolated from femurs and tibiae of 6~8 weeks old female C57BL/6 mice (Koatech, Pyungtaek, Gyeonggi, Korea). After lysing red blood cells, cells were incubated in minimal essential medium (Gibco BRL, Gaithersburg, MD, USA) supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 g/mL streptomycin in the presence of M-CSF (50 ng/mL) for 3 days. BMMs were obtained by removing floating cells. For osteoclast differentiation, BMMs (4 × 10 4 cells/well) were cultured in the presence of M-CSF (50 ng/mL) and RANKL (100 ng/mL) in 96-well plates with or without compounds (1)(2)(3)(4)(5). After 4 days, cells were fixed with 10% formalin for 5 min, stained for TRAP-positive cells, and photographed under a light microscopy. Quantitation of TRAP activity in culture supernatants was performed using TRAP staining kit (Kamiya Biomedical Company, Tukwila, WA, USA) according to the manufacturer's instructions.

Conclusions
We have isolated a new α-pyrone merosesquiterpenoid containing an angular tetracyclic carbon skeleton, ochraceopone F (1), and four known compounds, aspertetranone D (2), cycloechinulin (3), wasabidienone E (4), and mactanamide (5). All compounds had no cytotoxic effect on human cancer cell lines (HCT 15, NUGC-3, NCI-H23, ACHN, PC-3, and MDA-MB-231) up to 30 µg/mL. Aspertetranone D (2) and wasabidienone E (4) had a weak osteoclast differentiation inhibitory activity. Interestingly, mactanamide (5) displayed a potent suppression effect of osteoclast differentiation without any evidence of cytotoxicity at the effective concentrations. Mactanamide (5), a new fungistatic diketopiperazine containing the uncommon amino acid D-2,6-dihydroxyphenylalanine, has been first isolated from the mycelium of an undescribed marine fungus of the genus Aspergillus sp. from the surface of the brown marine alga Sargassum sp. [22]. However, to the best of our knowledge, this is the first report to describe the suppressive effect of mactanamide on osteoclast differentiation. This study revealed that mactanamide may have a significant role in the inhibition of osteoclast differentiation. However, to address the potential therapeutic efficacy, further research including mechanism of action study is needed.