Two New Alkaloids and a New Butenolide Derivative from the Beibu Gulf Sponge-Derived Fungus Penicillium sp. SCSIO 41413

Marine sponge-derived fungi have been proven to be a prolific source of bioactive natural products. Two new alkaloids, polonimides E (1) and D (2), and a new butenolide derivative, eutypoid F (11), were isolated from the Beibu Gulf sponge-derived fungus, Penicillium sp. SCSIO 41413, together with thirteen known compounds (3–10, 12–16). Their structures were determined by detailed NMR, MS spectroscopic analyses, and electronic circular dichroism (ECD) analyses. Butenolide derivatives 11 and 12 exhibited inhibitory effect against the enzyme PI3K with IC50 values of 1.7 μM and 9.8 μM, respectively. The molecular docking was also performed to understand the inhibitory activity, while 11 and 12 showed obvious protein/ligand-binding effects to the PI3K protein. Moreover, 4 and 15 displayed obvious inhibitory activity against LPS-induced NF-κB activation in RAW264.7 cells at 10 µM.


Introduction
The Beibu Gulf, a semi-closed gulf located in the northwest of the South China Sea, is rich in fishery or marine biological resources, including seagrass, coral, and sponge [1]. It has become an important source region of marine natural compounds [2]. The marine sponge, one of the most primitive and inferior multicellular animals, possesses abundant microorganisms germinated in its body and surface on account of its unique filter feeding system. Sponge-derived microorganisms act as an important guarantee for sponge survival since the lack of morphological physical defense structures [3][4][5].
In recent years, sponge-derived microorganisms have become one of the most abundant sources of new natural products, including terpenoids, alkaloids, sterols, peptides fatty acids, amino acids, and so on. Plenty of new secondary metabolites of sponge-derived fungi have been discovered with striking bioactive properties such as anti-tumor, antibacterial, antiviral, anti-inflammatory, and other biological activities [6]. Diketopiperazines are common secondary metabolites from a wide range of fungi, while quinazoline-containing diketopiperazines are relatively rare, with the difficulty in the determination of their configurations [7]. Butenolide derivatives, possessing the α,β-unsaturated γ-butyrolactone skeleton, were frequently isolated from fungi with diverse biological activities, especially the antitumor activity with diverse mechanisms or targets [8].
In our research for novel bioactive natural products from marine sponge-derived fungi, the strain fungus Penicillium sp. SCSIO 41413 was isolated from a Callyspongia sp. of their configurations [7]. Butenolide derivatives, possessing the α,β-unsaturated γ-butyrolactone skeleton, were frequently isolated from fungi with diverse biological activities, especially the antitumor activity with diverse mechanisms or targets [8].

Results
The fungus Penicillium sp. SCSIO 41413 was fermented in rice medium, which was extracted with EtOAc to obtain crude extract after 30-day fermentation. Several chromatographic methods, including silica gel column and semi-preparative HPLC with octadecylsilyl (ODS) column, were used for isolation of these 16 compounds.
Compound 1 was obtained as a yellowish oil.

Results
The fungus Penicillium sp. SCSIO 41413 was fermented in rice medium, which was extracted with EtOAc to obtain crude extract after 30-day fermentation. Several chromatographic methods, including silica gel column and semi-preparative HPLC with octadecylsilyl (ODS) column, were used for isolation of these 16 compounds.
Because of the diverse biological activities reported with quinazoline-containing diketopiperazines and butenolide derivatives [7,8], further bioactivity screening is necessary, such as anti-tumor related enzymatic bioassay. Enzymes phosphatidylinositol 3kinase (PI3K) and 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFK-2/FBPase 3, PFKFB3), which played a significant role in the regulation of glycolysis in cancer cells as well as its proliferation and survival [21], were used for assessment of enzyme activities in our study. As a result, none of the compounds showed inhibition above 50% against PFKFB3 enzyme at 20 µM, while 11 and 12 displayed obvious inhibition against PI3K with IC 50 values of 1.7 µM and 9.8 µM, respectively.
To obtain an insight into the molecular interactions between 11 and 12 and PI3K, molecular docking analysis was carried out. Butenolide derivatives 11 and 12 expressed the interaction with PI3K protein (PDB ID: 1E7U) perfectly, and the docking scores were −11.688 and −8.863, respectively. KWT (molecule of wortmannin) is a ligand in 1E7U designated by the RCSB. As shown in Figure 4A-C, phenolic hydroxy groups of 11 formed hydrogen bonds with the active site residues THR887, TYR867, and ALA885, and the ester group also interacted with VAL882 by hydrogen bond. Meanwhile, phenolic hydroxy groups of 12 formed three hydrogen bonds with residues THR887, TYR867, and ASP950. These results provide us rational explanation of the interactions between butenolide derivatives 11 and 12 and PI3K, which provides valuable information for further development of PI3K inhibitors. PFKFB3), which played a significant role in the regulation of glycolysis in cancer cells as well as its proliferation and survival [21], were used for assessment of enzyme activities in our study. As a result, none of the compounds showed inhibition above 50% against PFKFB3 enzyme at 20 μM, while 11 and 12 displayed obvious inhibition against PI3K with IC50 values of 1.7 μM and 9.8 μM, respectively.
To obtain an insight into the molecular interactions between 11 and 12 and PI3K, molecular docking analysis was carried out. Butenolide derivatives 11 and 12 expressed the interaction with PI3K protein (PDB ID: 1E7U) perfectly, and the docking scores were −11.688 and −8.863, respectively. KWT (molecule of wortmannin) is a ligand in 1E7U designated by the RCSB. As shown in Figure 4A-C, phenolic hydroxy groups of 11 formed hydrogen bonds with the active site residues THR887, TYR867, and ALA885, and the ester group also interacted with VAL882 by hydrogen bond. Meanwhile, phenolic hydroxy groups of 12 formed three hydrogen bonds with residues THR887, TYR867, and ASP950. These results provide us rational explanation of the interactions between butenolide derivatives 11 and 12 and PI3K, which provides valuable information for further development of PI3K inhibitors. Nuclear factor-κB (NF-κB) is a protein complex that controls transcriptional DNA, cytokine production, and cell survival, and is an important intracellular nuclear transcription factor [22]. The over-activation or defect of NF-κB can lead to the abnormal expression of various target cell genes, which is related to the inflammatory changes of many human diseases such as rheumatoid arthritis and heart and brain diseases. Therefore, inhibiting the NF-κB signal transduction pathway by drugs may become a means to treat many inflammatory diseases [23]. Seven compounds (3-6, 14-16) were screened for their inhibitory activities of LPS-induced NF-κB activation in RAW264.7 at 10 μM, and four of them (3-4, and 14-15) were revealed with the activity of varying strength ( Figure 5). Compounds 4 and 15 showed significant inhibitory activity against LPS-induced NF-κB (p < 0.001). Nuclear factor-κB (NF-κB) is a protein complex that controls transcriptional DNA, cytokine production, and cell survival, and is an important intracellular nuclear transcription factor [22]. The over-activation or defect of NF-κB can lead to the abnormal expression of various target cell genes, which is related to the inflammatory changes of many human diseases such as rheumatoid arthritis and heart and brain diseases. Therefore, inhibiting the NF-κB signal transduction pathway by drugs may become a means to treat many inflammatory diseases [23]. Seven compounds (3-6, 14-16) were screened for their inhibitory activities of LPS-induced NF-κB activation in RAW264.7 at 10 µM, and four of them (3-4, and 14-15) were revealed with the activity of varying strength ( Figure 5). Compounds 4 and 15 showed significant inhibitory activity against LPS-induced NF-κB (p < 0.001).

General Experimental Procedures
The NMR spectra were obtained on a Bruker Avance spectrometer (Bruker, Billerica, MA, USA) operating at 500 and 700 MHz for 1 H NMR, and 125 and 175 MHz for 13 C NMR, using tetramethylsilane as an internal standard. High-resolution mass spectra were recorded on a Bruker miXis TOF-QII mass spectrometer (Bruker, Billerica, MA, USA). Optical rotations were measured on a PerkinElmer MPC 500 (Waltham, MA, USA) polarimeter. UV and ECD spectra were recorded on a Chirascan circular dichroism spectrometer (Applied Photophysics, Leatherhead Surrey, UK). The TLC and column chromatography (CC) were performed on plates precoated with silica gel GF254 (10-40 μm), and over silica gel (200-300 mesh) (Qingdao Marine Chemical Factory, Qingdao, China), Sephadex LH-20 (Amersham Pharmacia Biotech AB, Uppsala, Sweden), and semi-preparative HPLC using an ODS column (YMC-pack ODS-A, YMC Co., Ltd., Kyoto, Japan, 10 mm × 250 mm, 5 μm). All solvents employed were of analytical grade. The sea salt (Guangzhou Haili Aquarium Technology Company, Guangzhou, China) was a commercial product.

Fungal Strain
The fungal strain Penicillium sp. SCSIO 41413 was isolated from a sponge (Callyspongia sp.) sample which was collected near the Weizhou Island (Guangxi, China), Beibu Gulf of the South China Sea. This strain was stored on MB agar (malt extract 15 g, sea salt 10 g, H2O 1 L, PH 7.4-7.8) slants at 4 °C and deposited at Key Laboratory of Tropical Marine Bioresources and Ecology, Chinese Academy of Sciences. The ITS sequence region of the strain SCSIO 41413 was amplified by PCR, and rDNA sequencing showed that it shared significant homology to that of Penicillium. The rDNA sequence has 100% sequence identity to that of Penicillium polonicum (GenBank accession no. NR_103687.1), so it was designated as Penicillium sp. SCSIO 41413.

Fermentation and Extraction
Seed medium (malt extract 15 g, sea salt 10 g, distilled water 1000 mL) was inoculated with Penicillium sp. SCSIO 41413 and incubated at 25 °C for 72 h on a rotating shaker (180 rpm/s). The strain Penicillium sp. SCSIO 41413 was cultured in the flasks (×60) of rice medium (rice 200 g/flask, sea salt 7.0 g/flask, distilled H2O 200 mL/flask). These flasks were incubated statically at 25 °C under a normal day/night cycle. After 30 days, the rice medium was soaked in EtOAc (600 mL/flask), cut into small pieces, and sonicated for 20 min. Then, they were poured into fermentation vats, which were extracted with EtOAc four times and concentrated under reduced pressure to obtain a crude extract. The crude

General Experimental Procedures
The NMR spectra were obtained on a Bruker Avance spectrometer (Bruker, Billerica, MA, USA) operating at 500 and 700 MHz for 1 H NMR, and 125 and 175 MHz for 13 C NMR, using tetramethylsilane as an internal standard. High-resolution mass spectra were recorded on a Bruker miXis TOF-QII mass spectrometer (Bruker, Billerica, MA, USA). Optical rotations were measured on a PerkinElmer MPC 500 (Waltham, MA, USA) polarimeter. UV and ECD spectra were recorded on a Chirascan circular dichroism spectrometer (Applied Photophysics, Leatherhead Surrey, UK). The TLC and column chromatography (CC) were performed on plates precoated with silica gel GF254 (10-40 µm), and over silica gel (200-300 mesh) (Qingdao Marine Chemical Factory, Qingdao, China), Sephadex LH-20 (Amersham Pharmacia Biotech AB, Uppsala, Sweden), and semi-preparative HPLC using an ODS column (YMC-pack ODS-A, YMC Co., Ltd., Kyoto, Japan, 10 mm × 250 mm, 5 µm). All solvents employed were of analytical grade. The sea salt (Guangzhou Haili Aquarium Technology Company, Guangzhou, China) was a commercial product.

Fungal Strain
The fungal strain Penicillium sp. SCSIO 41413 was isolated from a sponge (Callyspongia sp.) sample which was collected near the Weizhou Island (Guangxi, China), Beibu Gulf of the South China Sea. This strain was stored on MB agar (malt extract 15 g, sea salt 10 g, H 2 O 1 L, PH 7.4-7.8) slants at 4 • C and deposited at Key Laboratory of Tropical Marine Bioresources and Ecology, Chinese Academy of Sciences. The ITS sequence region of the strain SCSIO 41413 was amplified by PCR, and rDNA sequencing showed that it shared significant homology to that of Penicillium. The rDNA sequence has 100% sequence identity to that of Penicillium polonicum (GenBank accession no. NR_103687.1), so it was designated as Penicillium sp. SCSIO 41413.

Fermentation and Extraction
Seed medium (malt extract 15 g, sea salt 10 g, distilled water 1000 mL) was inoculated with Penicillium sp. SCSIO 41413 and incubated at 25 • C for 72 h on a rotating shaker (180 rpm/s). The strain Penicillium sp. SCSIO 41413 was cultured in the flasks (×60) of rice medium (rice 200 g/flask, sea salt 7.0 g/flask, distilled H 2 O 200 mL/flask). These flasks were incubated statically at 25 • C under a normal day/night cycle. After 30 days, the rice medium was soaked in EtOAc (600 mL/flask), cut into small pieces, and sonicated for 20 min. Then, they were poured into fermentation vats, which were extracted with EtOAc four times and concentrated under reduced pressure to obtain a crude extract. The crude extract was suspended in MeOH and then partitioned with an equal volume of petroleum ether (PE) in order to remove the oil. At last, the MeOH solution was concentrated under reduced pressure to obtain a black crude extract (85.0 g).
Two human prostate cancer cell lines, PC-3 (androgen receptor negative) and 22Rv1 (androgen receptor positive), were used in the cytotoxicity tests, and cell viability was analyzed by MTT assay as previously described [20].
The inhibitory activities of LPS-induced NF-κB activation in RAW264.7 cells were evaluated as detected by luciferase reporter gene assay as described previously [20]. In other words, the RAW264.7 cells stably transfected with luciferase reporter gene were placed in 96-well plates and pretreated with tested compounds (10 µM) and BA Y11-7082 (NF-κB inhibitor as positive control, 5 µM, Sigma-Aldrich) for 30 min. Then, they were stimulated with 5 µg/mL LPS for 8 h. The cells were collected, and luciferase activities of the triplicate tests were measured using the luciferase assay system (Promega, Madison, WI, USA).

Molecular Docking
The Schrödinger 2017-1 suite (Schrödinger Inc., New York, NY, USA) was employed to perform the docking analysis. The crystal structure of PI3K (PDB code: 1E7U) [26] obtained from the Protein Data Bank was used as a starting model with all of the waters and the N-linked glycosylated saccharides removed and was constructed following the Protein Prepare Wizard workflow in Maestro 11-1. The prepared ligands were then flexibly docked into the receptor using the induced-fit module with the default parameters. The figures were generated using PyMol molecular graphics software (Schrödinger 2017-1, Schrödinger Inc., New York, NY, USA).

Conclusions
In summary, chemical investigation of the marine fungus Penicillium sp. SCSIO 41413, which was isolated from a Beibu Gulf sponge sample, led to the isolation of two new alkaloids, polonimides E (1) and D (2), a new butenolide derivative, eutypoid F (11), and thirteen known compounds (3−10, 12−16). Their planar structures and absolute configurations were elucidated by detailed NMR, MS spectroscopic analyses, and measured and calculated ECD analyses. Butenolide derivatives 11 and 12 exhibited obvious inhibitory against the enzyme PI3K with IC 50 values of 1.7 µM and 9.8 µM, respectively, while 4 and 15 exhibited obvious inhibitory activity against LPS-induced NF-κB activation in RAW264.7 cells at 10 µM. The molecular docking with PI3K protein was also performed to understand the inhibitory activity. This study provides valuable information for further development of PI3K or NF-κB inhibitors.