Heterocornols from the Sponge-Derived Fungus Pestalotiopsis heterocornis with Anti-Inflammatory Activity

One strain-many compounds (OSMAC) manipulation of the sponge-derived fungus Pestalotiopsis heterocornis XWS03F09 resulted in the production of new secondary metabolites. The chemical study of the fermentation, cultivated on 3% artificial sea salt in the rice media, led to the isolation of twelve compounds, including eight new polyketide derivatives, heterocornols Q–X (1–8), one new ceramide (9), and three known analogues (10–12). The structures and absolute configurations of the new compounds were elucidated by spectroscopic data and calculated ECD analysis. Heterocornols Q (1) and R (2) are novel 6/5/7/5 tetracyclic polyketide derivatives featuring dihydroisobenzofuran and benzo-fused dioxabicyclo [4.2.1] nonane system, which might be derived from the acetyl-CoA by epoxidation, polyene cyclization, and rearrangement to form the core skeleton. Compound 12 showed moderate or weak antimicrobial activities against with MIC values ranging from 25 to 100 μg/mL. Heterocornols T and X (7 and 8) could inhibit the production of LPS-induced NO significantly, comparable to dexamethasone. Further Western blotting analysis showed 7 and 8 markedly suppressed the iNOS protein expression in LPS-induced RAW 264.7 cells in a dose-dependent manner. The result showed that 7 and 8 might serve as potential leads for development of anti-inflammatory activity.


Introduction
During the past several decades, marine-derived fungi are recognized as an important source of novel drug leads. However, most of their biosynthetic gene clusters are silent under laboratory conditions fungi, whereas only a fraction of gene clusters have been transcribed. In order to enlarge the diversity of metabolites, different strategies such as the one strain-many compounds (OSMAC) strategy [1][2][3][4][5], epigenetic modification [6][7][8], and genome mining have been devoted to activating the silent gene clusters [9][10][11][12][13]. Among them, the OSMAC strategy represents a simple strategy involving the systematic alteration of culture conditions.
Our previous investigation on the marine sponge-derived fungus Pestalotiopsis heterocornis XWS03F09 has resulted in the discovery of heterocornols A-P [14,15], pestaloisocoumarins A and B, isopolisin B, and pestalotiol A [16]. For exploring the chemical diversity of microorganisms using the OSMAC strategy, we reinvestigated the secondary metabolites of the strain XWS03F09, with additional 3% artificial sea salt to solid rice medium. Further chemical exploration resulted in the isolation of eight new polyketide derivatives, heterocornols Q-X (1-8), and one new ceramide (9), together with three known analogues, tabolites of the strain XWS03F09, with additional 3% artificial sea salt to solid rice medium. Further chemical exploration resulted in the isolation of eight new polyketide derivatives, heterocornols Q-X (1-8), and one new ceramide (9), together with three known analogues, pestalone (10) [17], and pestalachlorides A and B (11 and 12) [18]. The isolated compounds (1-9 and 12) were evaluated for the cytotoxic, antimicrobial, and anti-inflammatory activities in vitro. Here, we report the details of the isolation, structure elucidation, and biological activities of these compounds.
Unfortunately, in the NOESY spectrum, NOE interactions could not be used to determine the different stereoscopic relationship between 3 and 4. The apparent difference between 3 and 4 are the opposite Cotton effects ( Figure 4) and the opposite optical rotations. To further assign the absolute configuration of 3 and 4, ECD calculations were performed (Figures 4, S57 and S58). According to the literature [22,23], the ECD band between planar structures [20]. Compared with 1, the main difference occurred at the configuration at C-8, C-10, C-11, and C-13, which was proved by the carbon chemical shifts of C-8 (∆δ C (−0.1 ppm), C-10 (∆δ C 0.0 ppm), C-11 (∆δ C −4.1 ppm), C-13 (∆δ C −1.1 ppm), and C-14 (∆δ C −8.1 ppm).The NOESY correlations of H-8/H 3 -14 (Figures 3 and S14) along with the NOESY correlations for H-13/H-11 revealed that H-8 and H 3 -14 were positioned on the opposite face relative to H-10 and H-11. On the basis of the above evidence and the presumed biosynthetic pathway, we suggest that the relative configuration of 2 was assigned as 8R, 10R, 11S, 13S. This assignment was further confirmed by the calculated ECD spectrum of (8R, 10R, 11S, 13S)-2, the result of which showed good accordance with the experimental one ( Figure 4). Thus, the structure of 2 was determined and named heterocornol R (2).
Compound 4 was isolated as a white amorphous solid and had the same molecular formula, C 21 H 30 O 6 , as 3 by HRESIMS data ( Figure S21). Detailed analyses of its NMR spectroscopic features implied that 3 and 4 had the same planar structures. Analysis of the 2D NMR spectra confirmed the structure of 4 (Figures 2 and S22-S27).
Unfortunately, in the NOESY spectrum, NOE interactions could not be used to determine the different stereoscopic relationship between 3 and 4. The apparent difference between 3 and 4 are the opposite Cotton effects ( Figure 4) and the opposite optical rotations. To further assign the absolute configuration of 3 and 4, ECD calculations were performed (Figures 4,S28 and S29). According to the literature [22,23], the ECD band between 260 and 285 nm is allied with the 1 L b transition of the aromatic chromophore. The region of the spectrum from 260 to 285 is diagnostic of the configuration of aromatic compounds. The experimental ECD spectrum for compound 3 displays positive Cotton effects at 278 nm and negative effects at 208 and 300 nm. The calculated ECD spectra for 3 with an R configuration at C-12 showed a negative Cotton effect between 280 and 320 nm. The CD spectrum of 4 showed negative (229 and 240 nm) and positive (300 and 208 nm) Cotton effects, indicating that the C-12 of 4 was represented as S. As a result, the calculated ECD curve for 2R, 3R, 12R-3 was the best match with 3, and 2R, 3S, 12S-4 was the best match with 4 ( Figures 4 and S30-S35).
The molecular formula of 5 was assigned to be C 14 H 18 O 5 on the basis of the HRESIMS ion at m/z 289.1047 [M + Na] + and 13 C NMR data ( Figure S36). The 1 H and 13 C NMR data of 5 ( Figures S37-S39) were similar to those of vaccinol O [19]. The major difference was due to the presence of signals of the acetyl moiety (δ H 2.04, δ C 172.5, and 21.2) in 5, instead of the carbonyl group in vaccinol O. In addition, signals for an isopentyl unit were absent in the NMR spectra of 5 (Tables 2 and 3), which was confirmed by COSY and HMBC experiments ( Figures S40-S42). In addition, the CD spectrum of 5 exhibited a negative Cotton effect at 204 nm in accordance with that of vaccinol O [19], which allowed assignment of the 3R absolute configuration (Figure 4). The coupling constant J H-11/H-12 = 4.2 Hz revealed that the relative configurations of C-11 and C-12 in 5 were suggested as an erythro [24]. In order to discriminate between (11R, 12S)-5 and (11S, 12R)-5, the electronic circular dichroism (ECD) spectrum of 5 was calculated and compared with the experimental spectra. As a result, the calculated spectrum of (11R, 12S)-5 matched well with the experimental data (Figure 4), indicating absolute configuration of 5, to be 3R, 11R, 12S. 1.58, brs a NMR data for compounds 5-8 in CD 3 OD, b NMR data for compound 9 in CDCl 3 (δ in ppm and J in Hz).
The isolated compounds (1-9 and 12) were evaluated for their cytotoxic activities against four human cancer cell lines (Hela, A549, HCT-8, A2780) via MTT assay. However, none of them showed obvious cytotoxicities. They were also tested with their antimicrobial activities against three bacteria and one fungi using a micro broth dilution method (Table 4). Only compound 12 showed antibacterial activities against Gram-positive bacteria Staphylococcus aureus and Bacillus subtilis and Gram-negative bacteria Escherichia coli, with MIC values ranging from 25 to 50 μg/mL. Moreover, 12 also exhibited weak antifungal activities against Candida albicans with MIC values 100 μg/mL.
Anti-inflammatory in vitro screening tests activities revealed that 7 and 8 could inhibit the production of LPS-induced NO in RAW 264.7 cells significantly ( Figure 6A,B) with no cytotoxicity, comparable to the positive drug dexamethasone (DXM, 33 μM). Further Western blotting analysis showed 7 and 8 markedly suppressed the iNOS protein expression in LPS-induced RAW 264.7 cells in a concentration-dependent manner ( Figure  6C,D). The result showed that the two new polyketide derivatives, heterocornols T and X (7 and 8), might serve as potential leads for development of anti-inflammatory activity. Polyketides are common biosynthetic precursor in the process of synthesizing aromatics and macrolides in microorganisms. Compounds 1-8 were formed via different polyketide precursors by polyketide synthases. Presumed polyketide precursors are considered to be biogenetically derived from acetyl-CoA by reduction, dehydration, oxygenation, and cyclization to form the core skeleton. Next, presumed polyketide precursors underwent reduction to form a vicinal diol (an intermediate), which could generate a dihydroisobenzofuran through reduction and dehydration. Finally, the dihydroisobenzofuran undergoes dehydration and nucleophilic addition to yielded 1 or 2 (Scheme 1), featuring a novel 6/5/7/5 tetracyclic polyketide derivatives.
The isolated compounds (1-9 and 12) were evaluated for their cytotoxic activities against four human cancer cell lines (Hela, A549, HCT-8, A2780) via MTT assay. However, none of them showed obvious cytotoxicities. They were also tested with their antimicrobial activities against three bacteria and one fungi using a micro broth dilution method (Table 4). Only compound 12 showed antibacterial activities against Gram-positive bacteria Staphylococcus aureus and Bacillus subtilis and Gram-negative bacteria Escherichia coli, with MIC values ranging from 25 to 50 µg/mL. Moreover, 12 also exhibited weak antifungal activities against Candida albicans with MIC values 100 µg/mL.
Anti-inflammatory in vitro screening tests activities revealed that 7 and 8 could inhibit the production of LPS-induced NO in RAW 264.7 cells significantly ( Figure 6A,B) with no cytotoxicity, comparable to the positive drug dexamethasone (DXM, 33 µM). Further Western blotting analysis showed 7 and 8 markedly suppressed the iNOS protein expression in LPS-induced RAW 264.7 cells in a concentration-dependent manner ( Figure 6C,D). The result showed that the two new polyketide derivatives, heterocornols T and X (7 and 8), might serve as potential leads for development of anti-inflammatory activity.

Fungal Material
The fungal strain XWS03F09 was isolated from the sponge Phakellia fusca, which was collected from the Xisha Islands of China in 2012. It was identified as Pestalotiopsis heterocornis XWS03F09 by analysis of its ITS region of the rDNA as described, which has been deposited in the GenBank database (accession no. JN943628.1). A voucher specimen (No. XWS03F09) was deposited in the School of Pharmacy, Southwest Medical University, Luzhou, Sichuan, China.

Fermentation, Extraction, and Isolation
The fungal strain Pestalotiopsis heterocornis XWS03F09 was grown at 28 • C without shaking for 36 days in 1000 mL conical flasks containing solid rice medium (each flask contained 200 g of rice, 6 g of artificial sea salt; 200 mL of distilled water, boiled in an autoclave for 20 min at 121 • C). The total of fermented rice cultures was extracted with EtOAc three times to afford 165 g of crude extract ( Figure S64).
The extract was subjected to silica gel column chromatography eluting with a gradient CH 2 Cl 2 -MeOH (30:1-0:100) to give 9 fractions based on TLC properties. Fraction 3 was separated by silica gel column chromatography eluting with petroleum ether-EtOAc

Antimicrobial Assay
A micro broth dilution assay as previously reported [32] was used to evaluate the MICs of 1-9 and 12 against three bacteria (Staphylococcus aureus ATCC 25923, Bacillus subtilis ATCC 6633, Escherichia coli ATCC 25922), and one fungi (Candida albicans MYA-2867). The MIC was defined as the lowest concentration of the antimicrobial agent that completely inhibited visual growth of an organism. Ciprofloxacin and amphotericin B (Sigma Inc.) were used as positive controls against bacteria and fungi, respectively.

Western Blot Analysis
The RAW 264.7 cells were pretreated with compounds 7 (0, 3, 11, 33 µM), 8 (0, 3, 11, 33 µM), and DXM (33 µM) for 2 h, and then stimulated with LPS (1 µg/mL) for 24 h. Total protein was extracted via RIPA (Beyotime, Beijing, China) and the concentration measured by BCA protein assay kit (Beyotime, Beijing, China). The (40 µg) protein was separated with 10% SDS-PAGE and transferred onto PVDF membrane (Millipore, Billerca, MA, USA), which was blocked for 1 h with 5% non-fat milk in TBS at room temperature. Then, the membrane was incubated with primary antibody overnight, then washed three times with TBST and incubated with horseradish peroxidase conjugated secondary antibody for 1 h at room temperature, washed three times with TBST and visualized by CEL (Millipore). GAPDH served as an internal control. Band pattern was analyzed with Fluor Chem FC3 system (ProteinSimple, San Francisco, CA, USA).

Data Analysis
The data were expressed as the mean ± S.E. of at least three independent experiments. The statistical significance of the differences between the means was determined either using Student's t-test or one-way analysis of variance where appropriate. If the means were found to be significantly different, multiple pairwise comparisons were carried out by Tukey's post hoc test. The threshold value for acceptance of difference was 5% (p ≤ 0.05).

Conclusions
Eight new polyketide derivatives, heterocornols Q-X (1-8), one new ceramide (9), and three known analogues (10)(11)(12) were isolated from the sponge-derived fungus Pestalotiopsis heterocornis XWS03F09. The structures and absolute configurations of the new compounds were elucidated by spectroscopic data and calculated by ECD analysis. Compound 12 displayed growth inhibition towards S. aureus, B. subtilis, and E. coli, with MIC values ranging from 25 to 100 µg/mL. Heterocornols T and X (7 and 8) could inhibit the production of LPS-induced NO significantly, comparable to dexamethasone. The result showed that 7 and 8 might serve as potential leads for development of anti-inflammatory activity.