New Ophiobolins from the Deep-Sea Derived Fungus Aspergillus sp. WHU0154 and Their Anti-Inflammatory Effects

Deep-sea fungi have become a new arsenal for the discovery of leading compounds. Here five new ophiobolins 1–5, together with six known analogues 6–11, obtained from a deep-sea derived fungus WHU0154. Their structures were determined by analyses of IR, HR-ESI-MS, and NMR spectra, along with experimental and calculated electronic circular dichroism (ECD) analysis. Pharmacological studies showed that compounds 4 and 6 exhibited obvious inhibitory effects on nitric oxide (NO) production induced by lipopolysaccharide (LPS) in murine macrophage RAW264.7 cells. Mechanical study revealed that compound 6 could inhibit the inducible nitric oxide synthase (iNOS) level in LPS-stimulated RAW264.7 cells. In addition, compounds 6, 9, and 10 could significantly inhibit the expression of cyclooxygenase 2 (COX 2) in LPS-induced RAW264.7 cells. Preliminary structure-activity relationship (SAR) analyses revealed that the aldehyde group at C-21 and the α, β-unsaturated ketone functionality at A ring in ophiobolins were vital for their anti-inflammatory effects. Together, the results demonstrated that ophiobolins, especially for compound 6, exhibited strong anti-inflammatory effects and shed light on the discovery of ophiobolins as new anti-inflammatory agents.


Introduction
Deep-sea fungi, living under extreme environmental conditions such as high salinity, intensely high pressure, absence of sunlight, and deficiency of nutrients, are considered to be a new reservoir for drug discovery. Recently, a multitude of structurally unique and diverse natural products with promising pharmacological activities have been discovered from deep-sea fungi, which has attracted more attention of researchers for exploring new lead compounds from this type of extreme-environment microorganism [1][2][3][4][5]. Ophiobolins are a group of sesterterpene compounds which are characterized environment microorganism [1][2][3][4][5]. Ophiobolins are a group of sesterterpene compounds which are characterized by an intricate 5-8-5 fused ring system. Since Orsenigo isolated ophiobolin A from Bipolaris oryzae as a phytotoxin in 1957, around 70 ophiobolin analogues have been discovered from different fungi up to now [6][7][8][9][10][11][12]. Ophiobolins showed a broad spectrum of biological activities including anti-tumor [7,10,11,13], antimicrobial activity [14], anti-inflammatory [12], and calmodulin inhibitory effects [15]. Ophiobolins have attracted more attention of synthetic chemists and pharmacologists due to their structurally and biologically diversity, which show great promising for the development new agents for human diseases treatment [16].
As part of our ongoing efforts to search for new bioactive metabolites from marine microorganisms [17,18], we cultured a nonsporulating deep-sea derived fungus WHU0154. Eleven ophiobolins (compounds 1-11), including five new analogues 1-5, were obtained from the ethyl acetate (EtOAc) extract of the fungus WHU0154. Here we report the isolation and structural elucidation of the new compounds. Meanwhile, the anti-inflammatory activities of these isolated compounds were evaluated and the structure-activity relationship (SAR) was discussed.
The key HMBC correlations of H-8 (δ H 7.00) to C-21 (δ C 172.0), C-6 (δ C 52.9) and C-7 (δ C 130.6) confirmed that the aldehyde in asperophiobolin G (10) was oxidized to a carboxylic acid in 1. The key 1 H-1 H COSY and HMBC correlations confirmed the planar structure of 1 ( Figure 2 13 C-NMR data of 1 closely resembled those of asperophiobolin G (10) [12], with the presence of a carbonyl signal at δC 172.0 in 1 instead of an aldehyde signal at δC/H 194.7/9.24 seen in 10. The key HMBC correlations of H-8 (δH 7.00) to C-21 (δC 172.0), C-6 (δC 52.9) and C-7 (δC 130.6) confirmed that the aldehyde in asperophiobolin G (10) was oxidized to a carboxylic acid in 1. The key 1 H-1 H COSY and HMBC correlations confirmed the planar structure of 1 ( Figure 2). The relative configuration of 1 was deduced from a ROESY experiment ( Figure 3) as well as by comparing the NMR data with that of 10. The ROESY correlations of H-2 (δH 2.88) with H3-22 (δH 0.99), H-1β (δH 2.16) and of H-6 (δH 3.44) with H-1α (δH 1.20) indicated the trans-fused A/B ring which was confirmed by the 13 C resonance of C-1 at δC 47.1 and C-22 at δC 23.1 since the 13 C resonance signals of C-1 and C-22 usually resonate at higher field when the A/B ring are cis rather than trans [7].  [12,19]. On the basis of the empirical helicity rule relating the sign of the Cotton effect of the diagnostic O-C-C-O moiety, the negative Cotton effect at 310 nm indicated an 18R configuration. Finally, the absolute configuration of compound 1 was determined by experimental and calculated electronic circular dichroism (ECD) analysis ( Figure 4). Thus, the structure of compound 1 was determined and named 18,19-dihydro-18,19-dihydroxyasperophiobolin E. Full assignments of the 1 H-and 13 C-NMR data were achieved by analyses of 1D-and 2D-NMR spectra ( Table 1, Supplementary Figure S1-S10). The relative configuration of 1 was deduced from a ROESY experiment ( Figure 3) as well as by comparing the NMR data with that of 10. The ROESY correlations of H-2 (δ H 2.88) with H 3 -22 (δ H 0.99), H-1β (δ H 2.16) and of H-6 (δ H 3.44) with H-1α (δ H 1.20) indicated the trans-fused A/B ring which was confirmed by the 13 C resonance of C-1 at δ C 47.1 and C-22 at δ C 23.1 since the 13 C resonance signals of C-1 and C-22 usually resonate at higher field when the A/B ring are cis rather than trans [7].  4 in DMSO solution [12,19]. On the basis of the empirical helicity rule relating the sign of the Cotton effect of the diagnostic O-C-C-O moiety, the negative Cotton effect at 310 nm indicated an 18R configuration. Finally, the absolute configuration of compound 1 was determined by experimental and calculated electronic circular dichroism (ECD) analysis ( Figure 4). Thus, the structure of compound 1 was determined and named 18,19-dihydro-18,19-dihydroxyasperophiobolin E. Full assignments of the 1 H-and 13 C-NMR data were achieved by analyses of 1D-and 2D-NMR spectra ( Table 1, Supplementary Figure S1-S10).
Compound 2 was obtained as a white amorphous powder. The molecular formula was deduced as C 25 H 34 O 3 on the basis of HR ESI-Q-TOF MS and 13 C-NMR data, indicating nine degrees of unsaturation. The 1 H-NMR spectrum provided the resonances for five methyl groups and four olefinic protons, while the 13 C-NMR spectrum exhibited two carbonyl carbons for keto at δ C 196.7 and carboxylic acid group at δ C 174.7 and eight olefinic carbons for four double bonds. Comparison of the NMR data with those of asperophiobolin E (7) [12] revealed that they had similar structures, establishing an ophiobolin-based sesterterpenoid nucleus. The double bond at C-6 and C-7 in 2 was identified by HMBC correlations from H-2 (δ H 3.37) to C-6 (δ C 140.8) and from H-8 (δ H 2.52, 2.40) to C-6 (δ C 140.8), C-7 (δ C 139.5) and C-21 (δ C 174.7). Thus, the planar structure of 2 was determined, which was confirmed by key 1       Compound 3 was obtained as a white amorphous powder and had a molecular formula of C 25 H 34 O 4 , as determined by the HR ESI-Q-TOF MS and 13 C-NMR data, requiring nine indices of hydrogen deficiency. The 1 H-and 13 C-NMR spectra showed the characteristic signals of ophiobolin sesterterpenoid: five methyl groups at δ H/C 1.40/ 25.5, δ H/C 1.19/26.3, δ H/C 1.17/ 24.9, δ H/C 1.01/ 21.5, and δ H/C 0.90/ 23.7, eight olefinic carbons for four double bonds, and two carbonyl carbons for keto at δ C 198.0 and carboxylic acid group at δ C 175.0. Comparison of the 1 H-and 13 C-NMR data with those of 2 suggested that they had the similar structures and the difference between them lay in the chemical shift of C-8. The 13 C resonance of C-8 at δ C 73.4 in 3, but not δ C 32.3 in 2, indicated that compound 3 was a hydroxylated product of 2. The key 1 H-1 H COSY and HMBC correlations confirmed the deduction mentioned above (Figure 2). The relative configuration of compound 3 was identified by analysis of ROESY data and comparison of the NMR data with that of 2 ( Figure 3). The key ROESY correlation between H-8 (δ H 4.39) and H-10 (δ H 1.91) indicated that the hydroxyl group of C-8 was β orientation in 3. The Z-configuration of ∆ 16,17 was identified by the ROESY correlation of H-15 (δ H 2.64) and H-18 (δ H 6.09). The absolute configuration of compound 3 was determined by experimental and calculated electronic circular dichroism (ECD) analysis ( Figure 4). Thus, the structure of compound 3 was identified and named ∆ 16,17 -ophiobolin D. Full assignments of the 1 H-and 13 C-NMR data were achieved by analyses of 1D-and 2D-NMR spectra ( Table 1 Compound 4 was isolated as a white amorphous powder. It had the molecular formula of C 25 H 38 O 8 based on analyses of the HR ESI-Q-TOF MS and 13 C-NMR data, indicating seven degrees of unsaturation. The 1 H-and 13 C-NMR spectra showed great similarity to those of ophiobolin U [9], suggested that they had the similar structures. The difference between them was found in the NMR data of C-21. The 1 H and 13 C resonance of C-21 changed from δ 9.24/194.7 in ophiobolin U to δ C 174.7 in 4, indicating that the aldehyde group at C-21 in ophiobolin U was oxidized to carboxylic acid in 4. Thus, the planar structure of compound 4 was determined, which was confirmed by key 1 H-1 H COSY and HMBC correlations (Figure 2). The cis-fused A/B ring system was established by the key ROESY correlation of H-2 (δ H 2.31) with H-6 (δ H 3.17) and the 13 C resonance of C-1 at δ C 36.7 and C-22 at δ C 19.1 [7]. The other key ROESY correlations in compound 4 matched with those of ophiobolin U, indicating the same stereochemistry. The absolute configuration of compound 4 was determined by experimental and calculated electronic circular dichroism (ECD) analysis ( Figure 4). Thus, the structure of compound 4 was identified and named asperophiobolin L. Full assignments of the 1 H-and 13 C-NMR data were achieved by analyses of 1D-and 2D-NMR spectra ( Table 1 (Figure 3). The absolute configuration of compound 5 was determined by experimental and calculated electronic circular dichroism (ECD) analysis ( Figure 4). Thus, the structure of compound 5 was identified and named (16E)-asperophiobolin L. Full assignments of the 1 H-and 13 C-NMR data were achieved by analyses of 1D-and 2D-NMR spectra ( Table 1, Supplementary Figure S41-S50).

Anti-Inflammatory Bioactivities
The anti-inflammatory effects of ophiobolins 2-11 were first evaluated by detecting the NO production induced by LPS in murine macrophage RAW264.7 cells. Curcumin was used as positive control. Results showed that compounds 4 and 6 exhibited obvious inhibitory effects on NO production induced by LPS in RAW264.7 cells although no statistical significance was observed  Figure 5A). Meanwhile, these compounds except 6 had no cytotoxicity at all toward RAW264.7 cells at indicated concentrations ( Figure 5B). Even though compound 6 reduced the cell survival rate to~77 ± 18%, there was no statistical difference between control and compound 6 ( Figure 5B). Therefore, these compounds did not significantly affect cell survival.

Anti-Inflammatory Bioactivities
The anti-inflammatory effects of ophiobolins 2-11 were first evaluated by detecting the NO production induced by LPS in murine macrophage RAW264.7 cells. Curcumin was used as positive control. Results showed that compounds 4 and 6 exhibited obvious inhibitory effects on NO production induced by LPS in RAW264.7 cells although no statistical significance was observed ( Figure 5A). Meanwhile, these compounds except 6 had no cytotoxicity at all toward RAW264.7 cells at indicated concentrations ( Figure 5B). Even though compound 6 reduced the cell survival rate to ~77 ± 18%, there was no statistical difference between control and compound 6 ( Figure 5B). Therefore, these compounds did not significantly affect cell survival. Mechanical sudies revealed that compound 6 could obviously inhibit the expression of iNOS induced by LPS in RAW264.7 cells (Figure 6A-C). Also, compounds 6, 9, and 10 could inhibit the expression of COX 2 induced by LPS in RAW264.7 cells (Figure 6A,B). Preliminary structure-activity relationship (SAR) analysis revealed that the aldehyde group at C21 in ophiobolins is vital for their anti-inflammatory activities since compounds 1-3 and 7 didn't show obvious bioactivities. Also, α,βunsaturated ketone in A ring may also be crucial for their anti-inflammatory effects since compound 11 didn't exhibit obvious anti-inflammatory effect. Mechanical sudies revealed that compound 6 could obviously inhibit the expression of iNOS induced by LPS in RAW264.7 cells (Figure 6A-C). Also, compounds 6, 9, and 10 could inhibit the expression of COX 2 induced by LPS in RAW264.7 cells (Figure 6A,B). Preliminary structure-activity relationship (SAR) analysis revealed that the aldehyde group at C21 in ophiobolins is vital for their anti-inflammatory activities since compounds 1-3 and 7 didn't show obvious bioactivities. Also, α,β-unsaturated ketone in A ring may also be crucial for their anti-inflammatory effects since compound 11 didn't exhibit obvious anti-inflammatory effect.  Together, our studies demonstrated that ophiobolin 6 could inhibit the NO production induced by LPS in RAW264.7 cells by suppressing the expression of iNOS. Also, compounds 6, 9, and 10 could exhibit anti-inflammatory effect by inhibiting the expression of COX 2. These results have broaden the application of ophiobolins.

Discussion
Ophiobolins are widely distributed in fungus secondary metabolites, produced by the pathogenic plant fungi [6,20,21], mangrove endophytic fungi [12,22], and fungi from marine sediments [7,21]. Here we first reported the discovery of ophiobolins from deep-sea fungus from around 3200 m depth of the South China Sea. Previous studies were mainly focus on the phytotoxin, anti-bacterial and anticancer activities of this kind of compounds [7,10,11,13,14,[20][21][22][23]. Scattered reports were involved in the anti-inflammatory effects of this type of compounds [12,24]. Aniko et al., reported that treatment of male Wistar rats with 1.0 mg/kg of ophiobolin A could promote systemic inflammation by elevating the concentration of interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) as well as the activity of heme oxygenase (HO) and myeloperoxidase (MPO) in plasma [24]. Cai et al., reported that some ophiobolins could inhibit the production of NO induced by LPS in RAW264.7 macrophage cells [12]. Our results were line with that reported by cai et al., The contradictory results may be explained by different models of assessment and treatment dose and time point.
In addition, here we first discussed the preliminary SAR analysis of the anti-inflammatory effects of ophiobolins and their underlying mechanisms. Combined with previous report [12], we can conclude that the aldehyde group at C-21 and the α,β-unsaturated ketone functionality at A ring in ophiobolins were indeed crucial for their anti-inflammatory effects. In addition, compound 6 only slightly affected the cell survive with no statistical significance was observed, which indicated us to assess its cytotoxicity while evaluating its anti-inflammatory. To sum up, our results provide critical information for further medicinal chemistry research of ophiobolins and help for the discovery of more potent anti-inflammatory agents.

Fungal Material and Identification
Strain WHU0154 was isolated from a deep sea sample at 117 • 51.41 E, 19 • 50.71 N and 3197 m depth, South China Sea, on Thayer-Martin agar (glucose 10 g, peptone 5 g, KH 2 PO 4 1 g, MgSO 4 0.5 g, Rose Bengal 0.03 g, sea salts 15 g, agar 20 g, ddH 2 O 1L). The strain was first selected by antibacterial activities against Staphylococcus aureus ATCC 5165 of its culture crude extracts. It was identified as Aspergillus sp. according to the morphological characteristics of typical Aspergillus conidia structure (Supplementary Figure S51) and the internal transcribed spacer (ITS) sequence (GeneBank accession number MW228045), which is 98.85% similarity to Aspergillus asper NRRL 35910 (NCBI Reference Sequence: NR_151788.1), and is stored at −80 • C in School of Pharmaceutical Sciences, Wuhan University, China.

Cultivation, Extraction and Isolation
The initial cultures were maintained on the potato dextrose agar PDA (200 g of potato, 20 g of glucose, 20 g of agar, 1 L of pure water, pH natural) solid medium plates for 3 or 4 days. Matured spores were aseptically inoculated into 15 conical flasks (500 mL), each containing one-third of the PDA liquid medium (200 g of potato, 20 g of glucose, 1 L of pure water, pH natural), and continued to be cultured as seed solution for 7 days. Then the seed solution was aseptically inoculated into 500 mL/1L/3L conical flasks, each containing one-third of the liquid medium, the fermentation was performed on PDA liquid medium and statically cultured at 25 • C for 27 days.

Cell Culture
The murine macrophage RAW264.7 cells were obtained from the American Type Culture Collection (ATCC, Rockefeller, MD, USA). The cells were grown in Dulbecco's modified Eagle's medium (DMEM) medium with 10% FBS (ExCell bio, Shanghai, China) at 37 • C in a 98% humidified incubator with 5% CO 2 and 95% air. The cells were routinely split once every 1-2 days.

Measurement of Cell Viability
MTT assay was used to determine the effects of ophiobolins on the viability of RAW264.7 cells. All compounds were dissolved in DMSO at 10 mM stock concentration and stored at −20 • C. DMSO was used as blank control. The final concentration of DMSO kept below 0.1% in cell culture throughout the biological study. The cells were seeded in a 96-well plate (1.5 × 10 4 cells/well) overnight and treated with compounds (10 µM) and LPS (500 ng/mL) for 24 h. Then, 20 µL of MTT (5 mg/mL) was added to each well for an additional 4 h. The resulting formazan crystals after aspiration of the culture medium were dissolved in DMSO (150 µL/well) and the optical densities (OD, Synergy HT, BioTek, VT, USA) were measured at 570 nm. The data was presented as means ± SEM of three independent experiments.

NO Assay
The Griess method was used to measure NO concentrations in culture supernatants. Cells were seeded into 96-well plates at a density of 1.5 × 10 4 cells/well. After adhesion, the cells were treated with 10 µM of compounds and LPS (500 ng/mL) for 24 h. Nitrite release in the culture media was determined using the Griess reaction and presumed to reflect the NO levels. Briefly, the samples were mixed with equal volume of Griess reagent (1% sulfanilamide in 5% phosphoric acid and 0.1% naphthylethylenediamine dihydrochloride) and then incubated at room temperature for 10 min. The absorbance was measured at 540 nm on a microplate reader (Synergy HT, BioTek, VT, USA). And the NO concentration was determined at 540 nm using NaNO 2 as a standard. The data was presented as means ± SEM of three independent experiments

Western Blot
Briefly, 2 × 10 6 cells/well were seeded in a 6-well flat-bottomed plate, grown at 37 • C for 24 h, and treated with ophiobolin derivatives(10 µM) and LPS (100 ng/mL) for 24 h. Total cell lysates were harvested in lysis buffer (Beyotime Inst. Biotech, Shanghai, China) containing 1 mM phenylmethylsulfonyl fluoride (Beyotime Inst. Biotech). The protein concentration was measured using the Pierce ® BCA Protein Assay Kit (#23225, Pierce, Thermo, MA, USA). Equal amount of total proteins (~40 µg) were separated on 6-15% polyacrylamide gelelectrophoresis (SDS-PAGE) and transferred to poly(vinylidene fluoride) (PVDF) membranes (#IPVH00010, Millipore, Billerica, MA, USA). The membranes were blocked with 5% skim milk and probed with primary antibodies specific for iNOS, COX 2 and β-actin overnight at 4 • C followed by horseradish peroxidase conjugated secondary antibodies for 1 h, reacted with Pierce ® ECL Western Blotting Substrate (Thermo Fisher Scientific, Franklin, MA, USA) and detected by an ECL detection imaging system (BioTanon, Shanghai, China). The data was presented as means ± SEM of three independent experiments

Statistical Analysis
The statistical analyses were performed by using GraphPad Prism software version 5 (GraphPad Software, Inc., San Diego, CA, USA). Each experiment was performed at least three replicates and the results were presented as mean ± SEM. Multiple comparisons were carried out by one-way ANOVA, followed by Tukey's test. p < 0.05 was considered statistically significant.