Bioactive α-Pyrone Derivatives from the Endophytic Fungus Diaporthe sp. CB10100 as Inducible Nitric Oxide Synthase Inhibitors

Inducible nitric oxide synthase (iNOS) produces NO from l-arginine and plays critical roles in inflammation and immune activation. Selective and potent iNOS inhibitors may be potentially used in many indications, such as rheumatoid arthritis, pain, and neurodegeration. In the current study, five new compounds, including a dibenzo-α- pyrone derivative ellagic acid B (5) and four α-pyrones diaporpyrone A–D (9–12), together with three known compounds (6–8), were isolated from the endophytic fungus Diaporthe sp. CB10100. The structures of these new natural products were unambiguously elucidated using NMR, HRESIMS or electronic circular dichroism calculations. Ellagic acid B (5) features a tetracyclic 6/6/6/6 ring system with a fused 2H-chromene, which is different from ellagic acid (4) with a fused 2H-chromen-2-one. Both 2-hydroxy-alternariol (6) and alternariol (7) reduced the expression of iNOS at protein levels in a dose-dependent manner, using a lipopolysaccharide (LPS)-induced RAW264.7 cell models. Also, they decreased the protein expression levels of pro-inflammatory cytokines, such as tumor necrosis factor-α, interleukin-6 and monocyte chemotactic protein 1. Importantly, 6 and 7 significantly reduced the production of NO as low as 10 μM in LPS-induced RAW264.7 cells. Molecular docking of 6 and 7 to iNOS further suggests that both of them may interact with iNOS. Our study suggests that 6 and 7, as well as the alternariol scaffold may be further developed as potential iNOS inhibitors.


INTRODUCTION
Inflammation plays important roles in the occurring and development of many diseases, including rheumatoid arthritis (RA) (Choy and Panayi, 2001), osteoarthritis (OA) (Ahmad et al., 2020), diabetes (Purkayastha and Cai, 2015), and cancers (Zhong et al., 2016). When inflammation occurs, excessive inflammatory mediators are produced by inducible nitric oxide synthase (iNOS) or cyclooxygenase-2 (COX-2), such as NO and prostaglandin E2 (PGE 2 ) (Yu et al., 2019). Nonsteroidal anti-inflammatory drugs (NSAIDs) and steroid hormone glucocorticoids are both used for the treatment of inflammation, despite serious side effects (Buchman, 2001;Salvo et al., 2011). iNOS is a mammalian protein composed of a C-terminal reductase and an N-terminal oxygenase domain, which produces micromolar NO by oxidizing L-arginine to L-citrulline in the presence of bacterial lipopolysaccharide (LPS) and/or proinflammatory cytokines. A considerable number of iNOS inhibitors, such as arginine derivatives, pyrimidines and aminopyrimidines, as well as aliphatic, aromatic, and cyclic amidines, have been developed (Cinelli et al., 2020). Although many of them showed promise in the treatment of arthritis or inflammatory and neuropathic pain in animal models, there are no iNOS inhibitors on the market. Therefore, there is strong need to discover new iNOS inhibitors as anti-inflammation agents.
α-Pyrone (1, Figure 1A) is an aromatic unsaturated lactone and an important sub-structure of various natural products with interesting biological activities (Mcglacken and Fairlamb, 2005). For example, dothideopyrone F (2) inhibited NO production in LPS-induced BV2 cells and diminished the protein expression levels of iNOS and COX-2 (Kim et al., 2018). In addition, natural products with α-pyrone substructure have antibacterial (Zhang et al., 2013), antiinfluenza A virus (Hou et al., 2018), and anti-HIV (Appendino et al., 2007) activities. Some of them are signal molecules (Brachmann et al., 2013), while some are also cytotoxic agents against tumor cells (Rivera-Chávez et al., 2019;Zhu et al., 2019). In particular, dibenzo-α-pyrone (3) belongs to a significant group of heptaketide coumarin metabolites that have a fused tricyclic core ( Figure 1A), commonly identified from fungi, bacteria, and plants (Lai et al., 2016). Many of them also display diverse biological activities including cytotoxicity and anti-inflammatory activity (Mao et al., 2014). For example, ellagic acid (4), found in medicinal plants, vegetables and fruits, exhibits impressive anti-inflammatory and anti-diabetic activities (Ríos et al., 2018). It is widely used in skin care products because of its anti-oxidation effect and skin protection effects.
Sinomenine (Supplementary Figure  S1), a tetrahydroisoquinoline-type alkaloid isolated from the medicinal plant of Sinomenium acutum (Thunb.) ("青风藤" in Chinese), has strong anti-inflammatory effects and is used in China to treat RA for many years (Zhao et al., 2012;Wang et al., 2019). To our knowledge, there are few reports of endophytes and their natural products from S. acutum. Since endophytes from medical plants are attractive sources of bioactive natural products, we initiated an endeavor to isolate natural products from endophytes colonized in the stem and root of S. acutum, in our continuous search for new bioactive metabolites of microorganisms from un-or underexplored niches (Jiang et al., 2018;Jiang et al., 2021a). In this study, we report that these accumulating efforts have resulted in the isolation of multiple endophytes from S. acutum, and bioactivity-guided natural product isolation have yielded eight compounds, including a new dibenzo-α-pyrone derivative, ellagic acid B (5) and four new α-pyrone diaporpyrone A-D (9-12), together with three known compounds (6-8). Further biological evaluation of these compounds revealed that 2-hydroxy-alternariol (6) and alternariol (7) are potential iNOS inhibitors.

General Methods
The instruments (including those applied for optical rotations, MS, ORD, NMR, and ECD) and routine reagents for chemical isolation and biological evaluation were the same as those reported previously (Jiang et al., 2021a). The details were listed in the Supporting Information.

Isolation of Endophytes and Fermentation
The separation methods of endophytic fungi are detailed in the Supporting Information. These endophytic fungi CB10098-CB10104 were isolated from the stems and roots of S. acutum, which were collected from Huaihua (Hunan, China) in October 2017. They were grown in PDA (0.3% potato extract, 2% glucose, 1.5% agar power) medium at 28°C for 5 day, and then inoculated to seed medium [glucose 2%, sucrose 1%, soybean powder 0.2%, peptone 1%, K 2 HPO 4 0.03%, poly (ethylene glycol) 0.25%, NaNO 3 0.3%, and (NH 4 ) 2 SO 4 0.3%]. The final pH of the medium was adjusted to 6.0 before sterilization with an autoclave at 121°C for 20 min. Cultures were incubated in flasks at 28°C on a rotary shaker at 220 rpm for 3 day to prepare the seed culture. Next, they were transferred to 2 × 500 ml Erlenmeyer flasks contained rice (50 g) and H 2 O (50 ml). After sterilization in an autoclave at 121°C for 30 min, each flask was inoculated with 10 ml of seed culture and incubated at 28°C for 40 day. Finally, the solid rice culture containing the respective fungus were extracted by ethyl acetate to obtain extracts C1-C6.

Fungal Strain
The strain CB10100 was identified as Diaporthe sp. according to molecular identification and its ITS sequence has 99% sequence identity to that of Diaporthe sp. The sequence data have been deposited to GenBank with the accession number MW037206. The strain was deposited in the Xiangya International Academy of Translational Medicine, Central South University, Changsha, Hunan, China.

Large-Scale Fermentation of Diaporthe sp. CB10100
For large-scale fermentation of Diaporthe sp. CB10100, 185 × 1 L Erlenmeyer flasks containing rice (100 g) and H 2 O (100 ml) were used. The fermented solid rice culture was supersonically extracted with EtOAc (3 × 60 L), and after recovering the organic solvent, the crude extract was suspended in water and successively treated with petroleum ether, ethyl acetate, and n-butanol.

ECD Calculation Methods
The ECD spectrum of diaporpyrone B (10) was calculated by using of the Gaussian 09 package (Frisch et al., 2010). Those configurations were optimized at the B3LYP/6-31G (d) level. The ECD spectrum were calculated by the TDDFT method at the B3LYP/6-311+ +G (2 day, p) level with the CPCM model in methanol solution (Bruhn et al., 2013). The details were listed in the Supporting Information.

Cell Culture and Cell Viability Assay
Cell culture and cell viability assay (Liu et al., 2016) was utilized to investigate the maximum concentration of every compound that was not toxic to cells. The method for cell culture and cell viability assay was in the Supplementary Material.

Western Blotting for the Detection of iNOS and COX-2
The iNOS and COX-2 protein levels affected by compounds 5-12 were detected using Western blotting according to the reported procedures, and dexamethasone was used as the positive control (Jiang et al., 2021a). The procedure is detailed in the Supplementary Material.

Detection of NO and Inflammatory Cytokines Production
RAW264.7 cells were seeded in 12-well plates in a density of 2 × 10 5 cells/mL and incubated for 24 h. Three different concentration compounds were added in the each well for 1 h before LPS stimulation. The culture medium was collected and stored at −80°C. The levels of nitric oxide in the culture medium were detected using the Nitric oxide detection kit (Invitrogen) and the levels of TNF-α, IL-6 and MCP-1 were analyzed using the ELISA kits (eBioscience).

Molecular Docking Analysis
Molecular docking studies were conducted by the software Molecular Operating Environment (MOE 2010.06; Chemical Computing Group, Canada) as the computational platform. The procedure is detailed in the Supplementary Material.

Statistical Analysis
The results are expressed as mean ± SD of at least three independent experiments. Statistical significance of differences between groups was determined by ANOVA, and a level of *p < 0.05 or **p < 0.01 was considered statistically significant.

Isolation of Endophylic Fungi From Sinomenium acutum
Six endophytic fungi CB10098-CB10104 were isolated from the stems and roots of S. acutum, collected in Huaihua city in central China (Supplementary Figure S1). These endophytic microorganisms were fermented and the anti-inflammatory activity of their crude extracts C1-C6 was evaluated in a lipopolysaccharide (LPS)-stimulated RAW264.7 cells. Several crude extracts, e.g., C3 (from CB10100) and C5 (from CB10102), showed the specific inhibition against iNOS at a concentration of 20 μg/ml (Supplementary Figure S1C). Next, the fungus CB10100 was identified as a Diaporthe species based on DNA sequencing of its internal transcribed spacer 4 (ITS4) and the phylogenetic analysis of its ITS4 with selected Diaporthe strains in GenBank (Supplementary Figure S2). Diaporthe, the sexual morph of Phomopsis and a known plant pathogen (Huang et al., 2013), lives in a diversity of plants (Guarnaccia and Crous, 2017). Some Diaporthe or Phomopsis species can also produce H NMR ( a 500 MHz) or ( b 400 MHz) and 13 C NMR ( a 125 MHz) or ( b 100 MHz) data of 9-12 in CD 3 OD.  Figure S3). Therefore, a scale-up solid fermentation of Diaporthe sp. CB10100 (185 × 1 L flasks containing 100 g boiled rice) was next used to isolate natural products with anti-inflammatory activity.
Diaporpyrone A (9) was isolated as a colorless powder. Its molecular formula was determined as C 10  The 1 H and 13 C NMR data of 9 are similar to a previously isolated pyrone with a six-carbon aliphatic chain from an endophytic fungus Pericinia sp. F-31, with the replacement of the terminal methyl group by a carboxylic acid (δ C 178.1) (  Figure S36; Zhang et al., 2015). This was confirmed by the HMBC correlations from 1″ -CH 3 to C-5 (δ C 116.9) and C-6 (δ C 160.4), and from C-3 ʹ (δ C 34.4) to C-4 ʹ (δ C 176.8) and C-2 ʹ (δ C 26.2). We named the new α-pyrone 9 as diaporpyrone A, since it is from the fungus Diaporthe sp. CB10100.
Diaporpyrone D (12) was isolated as colorless powder, and its molecular formula was determined as C 8 H 8 O 4 by the (-)-HRESIMS m/z 167.0340 [M -H] − (calcd for C 8 H 7 O 4 , 167.0350), differing from the molecular formula of 9 by two methylenes. The UV maximum absorption and 1D NMR data were highly similar to those of 9 ( Table 2; Supplementary  Figures S4E,H), suggesting the presence of a similar carbon skeleton. The signals for 35.4 and 175.7 ppm are not present in the 13 C NMR spectrum of 12, while they are present in both HSQC and HMBC spectra (Supplementary Figures S69, S70). The complete structure of 12 was fully assigned based on these data. The complete structure of 12 was fully assigned based on 1 H− 1 H COSY, HSQC, and HMBC data ( Figure 2A; Table 2).

Biosynthetic Pathway Speculation for Compounds 5-12
The biosynthesis of 7 and 8 was proposed in 2012, while their biosynthetic enzyme SnPKS19 was identified from a wheat pathogen Parastagonospora nodorum (Kim et al., 2012;Chooi et al., 2015). Therefore, the biosynthesis of dibenzo-α-pyrone 5-8 in Diaporthe sp. CB10100 may follow the similar biosynthetic logic, which involves the condensation of seven molecules of malonyl-CoA, followed by aldol-type cyclization between C-2 and C-7, and C-8 and C-13, and the subsequent lactonization leads to the key intermediate 7 ( Figure 3A). Subsequent methylation of the C-5 hydroxyl group of 7 by a methyltransferase would lead to 8, and hydroxylation of C-2 of 7 may produce 6. Further multi-step modifications of 7 may be envisioned to generate the unique conjugated four ring system of 5, despite an alternative precursor of 5 may also be possible.
The biosynthesis of pyrone 9-12 may involve the condensation of three molecules of malonyl-CoA, followed by aldol-type cyclization and the subsequent lactonization to generate the key six-membered lactone intermediate by a PKS ( Figure 3B). Next, the additional fatty acid side chains with various lengths may be biosynthesized by a PKS and finally attached to the lactone to generate 9-12. The detailed biosynthetic mechanism of 9-12 remains to be elucidated.
Effects of Compounds 5-12 on LPS-Induced iNOS and COX-2 Expression in RAW264.7 Cells iNOS and COX-2 are key proteins in the inflammatory signaling pathway. When inflammation occurs, iNOS and COX-2 are usually highly expressed, resulting the formation of inflammatory mediators, including NO and PGE 2 (Kyoung et al., 2019). Many phenolic compounds exhibit antiinflammatory activities through inhibition of the iNOS (Ku et al., 2008;Laavola et al., 2015). No anti-inflammatory activity of compound 6 was reported (Pfeiffer et al., 2007;Chapla et al., 2014), while 7 was discovered to modify macrophage phenotype and to inhibit production of NO and inflammatory responses (Solhaug et al., 2015;Chen et al., 2020). In addition, dothideopyrone F (2) was able to diminish the expression of iNOS and COX-2 in lipopolysaccharide (LPS)induced BV2 cells (Kim et al., 2018). Therefore, we hypothesize that the newly isolated natural products, e.g., 5-6 and 9-12, may have certain anti-inflammatory activity. In lipopolysaccharide (LPS)-stimulated RAW264.7 cell model, we next evaluated their inhibitory activity of iNOS and COX-2 protein expression, along with 7 and 8, using dexamethasone (DEX) as a positive control ( Figures 4A-C).
Among the tested compounds, only 6 and 7 strongly inhibited iNOS protein expression, while they had no effects on COX-2 expression in a concentration of 30 μM ( Figure 4A). To compare the anti-inflammatory activity of 6 and 7, the anti-inflammatory activity of 6 and 7 in concentrations ranging from 10 to 40 μM was determined in LPS-stimulated RAW264.7 cells. Both compounds inhibit the expression of iNOS in a dosedependent manner (Figures 4D,E). For example, 6 was able to inhibit about 90% of iNOS expression when applied at 20 μM, while 7 showed slightly less inhibitory activity against iNOS expression. Therefore, the presence of multiple hydroxyl groups in the dibenzo-α-pyrone scaffold would be important for the observed inhibitory activity against iNOS expression, since the methylation of C-9 hydroxyl group abolishes the activity in 8, while the additional C-2 hydroxyl group increases the activity of 6. The fused α-pyran moiety into dibenzo-α-pyrone scaffold abolishes the inhibitory activity against iNOS expression activity in 5.
No significant cytotoxicity of both compounds to RAW264.7 cells was observed under the test conditions, even up to 100 μM of 6 and 7 were used ( Figures 4F,G). Solhaug et al. reported the cytotoxic activity of 7 in cells and we ascribe this difference as the Frontiers in Chemistry | www.frontiersin.org May 2021 | Volume 9 | Article 679592 6 shorter treatment period (18 h) in our study, compared to the previously used 48 h (Solhaug et al., 2015). These results suggest that the inhibitory activities of 6 and 7 against iNOS expression in LPS-stimulated RAW264.7 cells did not involve general cytotoxicity.
2-Hydroxy-alternariol (6) and Alternariol (7) Decreased the Protein Levels of TNF-α, IL-6 and MCP-1 in LPS-Stimulated RAW264.7 Cells Macrophages are central effectors in inflammation, and activated macrophages act through the release of cytokines, such as tumor necrosis factor α (TNF-α) and interleukin-6 (IL-6), as well as chemokines, such as monocyte chemoattractant protein-1 (MCP-1) (Fujiwara and Kobayashi, 2005). Since TNF-α, IL-6, and MCP-1 are major mediators of inflammation, we examined the effects of 6 and 7 on LPS-induced TNF-α, IL-6, and MCP-1 production in RAW264.7 cells, using DEX as a control (Figures 5A-F). After RAW264.7 cells were stimulated with LPS (100 ng/ml) for 18 h in the presence or absence of 6 and 7, the levels of TNF-α, IL-6, and MCP-1 in the culture media were measured by the ELISA. As shown in Figures 5A-C, compound 6 is able to inhibit the production of the tested cytokines and chemokines in a dosedependent manner in RAW264.7 cells stimulated with LPS (p < 0.05). Significantly, the protein level of IL-6 is reduced to ∼25% by 6 (40 μM), which is comparable to the treatment by DEX (0.5 μM). Similar inhibitory effects of 7 could be observed for TNF-α and IL-6, while intriguingly, higher concentrations of 7, e.g., 20 and 40 μM, have no obvious effects on MCP-1 production.
Anti-Inflammatory Effects of 2-Hydroxy-Alternariol (6) and Alternariol (7) on LPS-Induced NO Production in LPS-Stimulated RAW264.7 Cells NO, an important cellular messenger, may mediate various biological processes, including inflammation, induction of tumor cell death, and killing alien microorganisms (Jiang et al., 2021b). We next investigated the release of NO by the treatment of 6 and 7 in LPS-stimulated RAW264.7 cells (Figures 5G,H). The level of nitrite, a stable oxidized product of NO in the culture medium of RAW264.7 cells were measured using a nitric oxide detection kit. The treatment with 6 and 7 reduced LPS-induced NO production in a dose-dependent manner in the range of 10-40 μM. Compound 6 showed stronger inhibition of NO production than 7 in 10 μM, consistent with the more potent inhibition of iNOS by 6.

Molecular Docking Simulations of 6 and 7 With iNOS
Due to the reduction of NO level and the reduced expression of iNOS by the treatment of 6 and 7 in LPS-treated RAW264.7 cells, compounds 6 and 7 may interact with iNOS and block the biosynthesis of NO. To our knowledge, the dibenzo-α-pyrone Frontiers in Chemistry | www.frontiersin.org May 2021 | Volume 9 | Article 679592 scaffold has not been used as iNOS inhibitors. Therefore, the interactions of 6 and 7 with iNOS were investigated using molecular docking by Molecular Operating Environment (MOE) (Hu et al., 2017;Jiang et al., 2021b). Using the crystal structure of mouse inducible nitric oxide synthase (miNOS, 3NW2) as a template, compound 6 form four hydrogenbonding interactions with the amino acid residue Trp457, Asp376, and Arg382 of iNOS, while 7 only has one hydrogen-bonding interaction and two π-π interactions with Met428 ( Figures 5I,J). Taken together, 6 and 7 may interact with iNOS and could be further developed as specific inhibitors against iNOS.

CONCLUSION
In conclusion, five new compounds, including a new dibenzoα-pyrone derivative, ellagic acid B (5) and four new α-pyrone Frontiers in Chemistry | www.frontiersin.org May 2021 | Volume 9 | Article 679592 8 diaporpyrone A-D (9-12), together with three known compounds (6-8), were isolated from the endophytic fungus Diaporthe sp. CB10100. Their structures were determined by the analyses of their NMR and HRESIMS spectra, while the absolute configuration of 10 and 11 was based on ECD spectra and their specific optical rotations. Ellagic acid B (5) features a tetracyclic 6/ 6/6/6 ring system with a fused 2H-chromene, while diaporpyrones consist of a pyrone and a short fatty acid side chain. The biosynthesis of 5 and 8 may involve a fungus PKS similar to SnPKS19 from P. nodorum, while the biosynthesis of 9-12 would likely be catalyzed by distinct fungi PKSs. All of the isolated compounds 5-12 were evaluated for their antiinflammatory activities in LPS-stimulated RAW 264.7 macrophages. Among them, 6 and 7 display strong FIGURE 5 | Compounds 6 and 7 inhibit the production of inflammatory cytokines and NO, probably through iNOS. Effects of 6 (A-C) and 7 (D-F) on the protein expression levels of TNF-α, IL-6, and MCP-1 in RAW264.7 cells. The cells were seeded in 12-well plates in a density of 2 × 10 5 cells/mL, incubated for 18 h and their protein expression were analyzed using the ELISA kit (eBioscience). Effects of 6 (G) and 7 (H) on the production of NO in RAW264.7 cells. The cells were pretreated for 1 h with the indicated concentrations of 6 or 7 and then stimulated with LPS (100 ng/ml) for 24 h. NO production in the culture medium was detected using NO detection kit (Invitrogen). The data shown represent the mean values of three independent experiments. *p < 0.05, **p < 0.01. Molecular docking of 6 (I) and 7 (J) with iNOS (PDB ID: 3NW2) using MOE. For clarity, only interacting residues are labeled and hydrogen bonding interactions are shown by dashed arrows.
Frontiers in Chemistry | www.frontiersin.org May 2021 | Volume 9 | Article 679592 inhibitory activities against iNOS and inhibit nitric oxide production. In addition, both compounds significantly reduced the production of pro-inflammatory mediators and cytokines, including NO, TNF-α, IL-6, and MCP-1. Molecular docking of 6 and 7 to iNOS further suggests that they are potential iNOS inhibitors. Considering the importance of iNOS in inflammation and signal transduction, the discovery of 6 and 7 the alternariol scaffold may be further developed as potential iNOS inhibitors.

DATA AVAILABILITY STATEMENT
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary Material.

AUTHOR CONTRIBUTIONS
YH, YD, and HP conceived and designed the experiments. HP, WZ, BH, and CN was responsible for the isolation of compounds. YH and HP elucidated the structures. JL, YP, YT, and XH tested the anti-inflammation of the compounds. YW performed the experiments of docking. YH, YD, and HP interpreted the data and wrote the paper. All authors read and approved the final manuscript.