New Polyketides from Mangrove Endophytic Fungus Penicillium sp. BJR-P2 and Their Anti-Inflammatory Activity

Four new polyketide compounds, including two new unique isocoumarins penicillol A (1) and penicillol B (2) featuring with spiroketal rings, two new citreoviridin derivatives citreoviridin H (3) and citreoviridin I (4), along with four known analogues were isolated from the mangrove endophytic fungus Penicillium sp. BJR-P2. Their structures were elucidated by extensive spectroscopic methods. The absolute configurations of compounds 1–4 based on electronic circular dichroism (ECD) calculations, DP4+ analysis, and single-crystal X-ray diffraction are presented. All the new compounds were evaluated for anti-inflammatory activity. An anti-inflammatory assay indicated that compound 2 inhibited lipopolysaccharide (LPS)-induced NO production in RAW 264.7 cells, with half-maximal inhibitory concentration (IC50) values of 12 μM, being more potent than the positive control, indomethacin (IC50 = 35.8 ± 5.7 μM). Docking study showed that compound 2 was perfectly docking into the active site of murine inducible nitric oxide oxygenase (iNOS) via forming multiple typical hydrogen bonds.


Introduction
Inflammation is an adaptive response triggered by harmful stimuli, including some conditions of infection and tissue damage [1,2]. Macrophages, neutrophils, and lymphocytes are important natural immune cells to participate in homeostasis and immune response, and play complicated action on the pathogenesis of inflammatory disease [3][4][5]. Stimulated immune cells regulate inflammation by producing proinflammatory factors and mediators, such as interleukin (ILS), tumor necrosis factor-α (TNF-α), NO, prostaglandin E2 (PGE2), and iNOS [6][7][8]. Therefore, inhibition of inflammatory cytokines, chemokines, and mediators can be potent therapeutic strategies for the prevention of inflammationrelated diseases.
Penicillium species are among the most widespread fungal organisms on earth and contains more than 350 species. Many Penicillium species can produce plentiful secondary metabolites, such as alkaloids [9], polyketides [10], cyclic peptides [11], and terpenoids [12], that can ascribe specific structural characteristics and significant biological activities. Isocoumarins are important natural lactones with wide range of biological activities, such as neuroprotective, antibacterial, antivirus and antitumor activities, and distribute widely in various microorganisms and plants from natural sources [13][14][15][16][17][18][19]. Up to now, nearly 1000 naturally occurring isocoumarins were reported [14]. As our continuing interest in finding new compounds with potential anti-inflammatory activity, the chemical investigation of the endophytic fungus Penicillium sp. BJR-P2 yielded two new unique isocoumarins with spiroketal rings (1)(2), two new citreoviridin derivatives (3)(4), together with four known analogues (5)(6)(7)(8) (Figure 1). In this paper, the stereochemistry of these compounds was determined for the first time by DP4+ analysis, ECD calculations, and single-crystal

Structural Elucidation
Compound 1 was obtained as a white powder. Its molecular formula was determined to be C 15  3 Hz) were also recorded in this spectrum. The above spectroscopic features indicated that 1 belonged to the isocoumarin class. Further analysis of HMBC spectrum (Figure 2), the correlations from H-7 to C-2 (δ C 101.0)/C-5 (δ C 106.8)/C-6 (δ C 166.7)/C-8 (δ C 164.4), from H-5 to C-2/C-6, from H-4 to C-2/C-3 (δ C 140.2)/C-6' (δ C 104.6), from H-9 to C-6 suggested that 1 was an isocoumarin derivative with a hydroxyl group at C-8 and a methoxy group at C-6. The 1 H-1 H COSY correlations between H-2', H-3', H-4', H-5', and H-10 combined with the HMBC correlations from H-5' to C-3' (δ C 38.5)/C-4' (δ C 63.4), from H-3' to C-2' (δ C 63.0)/C-10 (δ C 20.5), from H-10 to C-2'/C-3' suggested an aliphatic fragment of -CH 2 -CH-CH 2 -CH-CH 3 . Furthermore, the key HMBC correlations from H-5' to C-1/C-4/C-6', from H-4' to C-6', and from H-10 to C-6', together with the unsaturation of compound 1 and the chemical shift of C6' (δ C 104.6) indicated the presence of the spiroketal ring C. Therefore, the planar structure of 1 was shown in Figure 1. The absolute configuration of 1 was further verified by the X-ray diffraction analysis of a single crystal using Cu Kα as 6'S, 2'S, 4'S-1 ( Figure 3). Hence, the structure of compound 1 was identified as 6 S, 2'S, 4 S-1 and named penicillol A. H-10 combined with the HMBC correlations from H-5' to C-3' (δC 38.5)/C-4' (δC 63.4), from H-3' to C-2' (δC 63.0)/C-10 (δC 20.5), from H-10 to C-2'/C-3' suggested an aliphatic fragmen of -CH2-CH-CH2-CH-CH3. Furthermore, the key HMBC correlations from H-5' to C-1/C 4/C-6', from H-4' to C-6', and from H-10 to C-6', together with the unsaturation of com pound 1 and the chemical shift of C6' (δC 104.6) indicated the presence of the spiroketa ring C. Therefore, the planar structure of 1 was shown in Figure 1. The absolute configu ration of 1 was further verified by the X-ray diffraction analysis of a single crystal using Cu Kα as 6'S, 2'S, 4'S-1 ( Figure 3). Hence, the structure of compound 1 was identified a 6′S, 2'S, 4′S-1 and named penicillol A.      , and one active proton at δ H 11.04 (s) were also recorded in this spectrum. Cumulative analyses of the 1 H and 13 C NMR spectra of compound 2 revealed that it possessed the similar planar structure as that of 1. The main difference was the change of hydroxylated methine in 1 (δ C 63.4) to a carbonyl in 2 (δ C 202.5), indicating that compound 2 was an analog of 1. Furthermore, the planar structure of 2 was further verified by the key HMBC correlation from H-5' to C-4' (δ C 202.5), and H-3' to C-4' (Figure 2). The absolute configuration of 2 was determined by comparison of experimental and calculated ECD. Experimental data showed that the compounds 1, 2, and 6 had extremely similar ECD spectra, inferring that the cotton effect of compound 2 might be only affected by the chiral centers of C-6' (δ C 104.7) and C-2' (δ C 67.9) ( Figure 4A). Then, the ECD spectra of the four possible configurations (6'S, 2'S-2; 6'S, 2'R-2; 6'R, 2'S-2; 6'R, 2'R-2) were calculated. The results disclosed that 6'S, 2'S-2 and 6'S, 2'R-2 displayed similar ECD spectra with the experimental one, showing a negative cotton effect (CE) at about 266 nm and a positive CE at about 285 nm ( Figure 4B). Therefore, it was rationally speculated that the cotton effect of compound 2 was only affected by the chiral center of C-6', and the configuration of C-6' was determined to be 6'S as same as that of compound 1. Based on the biosynthetic point of view, the absolute configuration at C-2 should be the same as that of compound 1. Therefore, the structure of compound 2 was identified as 6'S, 2'S-2 and named as penicillol B.
A plausible typical fungal polyketide synthetase (PKS) involved biosynthetic pathway for compounds 1-4 was proposed as shown in Figure S33. The condensation of one mole of acetyl coenzyme A and six moles of malonyl coenzyme A gives a mole linear polyketide chain. Subsequent keto-reduction, cyclization, methylation, and hydroxylation furnish compounds 1 and 2. Similarly, one mole acetyl coenzyme A and eight moles malonyl coenzyme A condensed to form a mole linear polyketide chain, then keto-reduction, dehydration, cyclization, methylation, and hydroxylation form compounds 3 and 4 [25,26].

Anti-Inflammatory Activity
New compounds 1-4 were evaluated for anti-inflammatory activity in LPS-stimulated RAW 264.7 macrophages. Especially, Compound 2 significantly inhibited nitric oxide production with an IC 50 value of 12 µM (Table 3). Table 3. Inhibitory activities against LPS-induced NO production.

Molecular Docking Studies
Inhibition of NO overproduction is usually the result of inhibition of iNOS enzyme expression or activity [27,28]. In order to investigate the inhibitory mechanism of compound 2 on NO production, the interaction and binding mode between compound 2 and iNOS (PDB: 3E6T) [29], molecular docking study was carried out using AUTODOCK 4.2.6 modeling software. Docking procedure was validated by docking of ligand indomethacin (positive drug) in the active site of iNOS, and root-mean square deviation (RMSD) of 0.12 Å to the X-ray structure. The results revealed that the lowest energy of compound 2 (−7.49 Kcal/mol) was lower than that of positive drug indomethacin (−7.45 Kcal/mol), and the lowest energy of compound 1 (−7.36 Kcal/mol) was higher than that of positive drug. Further observations showed indomethacin formed a hydrogen bond with key amino acid residues GLU-371, two hydrogen bonds with amino acid residues ARG-260, and a hydrogen bond with GLN-257 in the iNOS active pocket ( Figure 6A). Compound 2 formed a hydrogen bond with the key amino acid residue GLU-371 through the methoxy group, two hydrogen bonds with the residue ASP-379 and ARG-382 by the hydroxyl group in the iNOS active pocket ( Figure 6B), respectively. Notably, for compound 1, while the carbonyl group in compound 2 at the 4' position changed to the hydroxyl group, the optimized conformation of 1 was different from that of compound 2 and could not enter into the iNOS active pocket to form hydrogen bonds with the key amino acid residues ( Figure 6C). As a result, compound 1 had no production inhibition activity (Table 3).

Anti-Inflammatory Activity
New compounds 1-4 were evaluated for anti-inflammatory activity in LPS-stimulated RAW 264.7 macrophages. Especially, Compound 2 significantly inhibited nitric oxide production with an IC50 value of 12 μΜ (Table 3).

Molecular Docking Studies
Inhibition of NO overproduction is usually the result of inhibition of iNOS enzyme expression or activity [27,28]. In order to investigate the inhibitory mechanism of compound 2 on NO production, the interaction and binding mode between compound 2 and iNOS (PDB: 3E6T) [29], molecular docking study was carried out using AUTODOCK 4.2.6 modeling software. Docking procedure was validated by docking of ligand indomethacin (positive drug) in the active site of iNOS, and root-mean square deviation (RMSD) of 0.12 Å to the X-ray structure. The results revealed that the lowest energy of compound 2 (−7.49 Kcal/mol) was lower than that of positive drug indomethacin (−7.45 Kcal/mol), and the lowest energy of compound 1 (−7.36 Kcal/mol) was higher than that of positive drug. Further observations showed indomethacin formed a hydrogen bond with key amino acid residues GLU-371, two hydrogen bonds with amino acid residues ARG-260, and a hydrogen bond with GLN-257 in the iNOS active pocket ( Figure 6A). Compound 2 formed a hydrogen bond with the key amino acid residue GLU-371 through the methoxy group, two hydrogen bonds with the residue ASP-379 and ARG-382 by the hydroxyl group in the iNOS active pocket ( Figure 6B), respectively. Notably, for compound 1, while the carbonyl group in compound 2 at the 4' position changed to the hydroxyl group, the optimized conformation of 1 was different from that of compound 2 and could not enter into the iNOS active pocket to form hydrogen bonds with the key amino acid residues ( Figure  6C). As a result, compound 1 had no production inhibition activity (Table 3).

Fungal Material
The fungus BJR-P2 used in this research was isolated from the barks of Avicennia marinav (Forsk.) Vierh, a mangrove plant which collected from Yangjiang Hailing Island Mangrove Wetland Park. Using molecular biology methods, the fungus was identified through DNA amplification and ITS region sequencing. The sequence data of this strain has been deposited in Gen Bank with accession no. PRJNA793386. The BLAST search results show that the sequence is 100% similar to that of Penicillium sp.

ECD and 13 C NMR Calculation
The conformational searches of the compounds were carried out by means of the Spartan'14 software and at Molecular Merck force field (MMFF) and DFT/TD-DFT calculations. Furthermore, Gaussian 05 program was used to generate and optimize the conformer at B3LYP/3-21G (d) level. Conformers with a Boltzmann distribution of over 1% were chosen for optimization at B3LYP/6-31+G (d, p), meanwhile, ECD calculation were conducted with the TD-DFT method at the B3LYP/6-31+G (d, p) level and the 13 C NMR calculation at mPW1PW91-SCRF/6-311+g (2d, p). The ECD spectra were generated using the SpecDis 3.0 (University of Würzburg, Würzburg, Germany) and Origin Pro 8.0 (Origin Lab, Ltd., Northampton, MA, USA) from dipole length rotational strengths by applying Gaussian band shapes with sigma = 0.30 eV. Then, the calculated and theoretical values of 13 C were analyzed by DP4+ [30].

Anti-Inflammatory Assays
The RAW 264.7 mouse macrophage cell line was purchased from the Cell Bank of Shanghai Institute of Biochemistry and Cell Biology (Chinese Academy of Sciences, Shanghai, China). Murine macrophage RAW 264.7 cells were cultured in DMEM (high glucose) medium supplemented with 10% (v/v) fetal bovine serum, 100 µg·mL −1 penicillin and streptomycin, and 10 mM HEPES buffer at 37•C in 5% CO 2 in air for 1 h. Cells were pretreated with different concentrations of samples (10, 5, 2.5, 1.25, and 0.625 µM) dissolved in serum-free medium containing 0.5% DMSO for 4 h, followed by stimulation with 1 µg·mL −1 LPS for 24 h. A total of 50 µL cell culture medium was mixed with 100 µL Griess reagents I and II and incubated horizontally at room temperature for 10 min. The absorbance was measured at 570 nm [31,32].

Molecular Docking Study
Virtual docking is implemented in the AutoDock tool of AutoDock4.2.6 software [33]. This is a common docking method that allows the ligand to have sufficient flexibility and maintain the rigidity of the target protein. The X-ray crystal structure of iNOS (PDB ID: 3E6T) [27] was obtained from the RCSB protein database (PDB) database. Before docking simulation, PyMOL was used to delete the original ligand and water molecules from the crystal structure, and the protein was saved in PDB format (receptor.pdb). The compound structure was drawn using ChemDraw 2D software, which was converted into three-dimensional (3D) structure by ChemDraw 3D software, and then stored as a file in PDB format. Furthermore, the molecular structure was optimized by Gaussian software. AutoDock tools converted both protein and ligand into PDBQT format for subsequent docking. Focusing on the protein, the parameters of the grid box were set to 126 × 126 × 126 points and the Lamarckian genetic algorithm was used to link the algorithm with 100 GA operations. Finally, PyMOL was used to visualize and analyze the results.

Conclusions
In conclusion, chemical investigation of the mangrove endophytic fungus BJR-P2 resulted in the isolation and identification of four new compounds (1)(2)(3)(4), with four known analogs (5)(6)(7)(8). Their structures were elucidated by extensive spectroscopic methods and quantum chemical calculations. The anti-inflammatory activity evaluation was carried out by screening their inhibition activity on NO production. The results showed compound 2 exhibited significant inhibitory activity with an IC 50 value of 12 µM. This study may provide a new chemical lead candidate for the discovery of anti-inflammatory agents.