Indole diketopiperazine alkaloids and aromatic polyketides from the Antarctic fungus Penicillium sp. SCSIO 05705

Abstract A new indole diketopiperazine alkaloid, named penilline D (1), together with five known indole alkaloid analogues (2–5, 11), two meroterpenoids (6 and 12), and four butenolide derivatives (7–10), were isolated from the Antarctic fungus Penicillium sp. SCSIO 05705. Extensive spectroscopic analysis and electronic circular dichroism (ECD) calculation were used to elucidate the structure of penilline D (1), including its absolute configuration. All isolated compounds (1–12) were evaluated for their cytotoxic, antibacterial and enzyme inhibitory activities against acetylcholinesterase (AChE) and pancreatic lipase (PL). Among them, compound 5 exhibited moderate in vitro cytotoxic activity against the 143B cell line with IC50 value of 12.64 ± 0.78 μM. Compound 6 showed strong inhibitory activity against AChE with IC50 value of 0.36 nM (IC50 18.7 nM for Tacrine), while compounds 6 and 11 showed weak PL enzyme inhibitory activity. Furthermore, an in silico molecular docking study was also performed between 6 and AChE. Graphical Abstract


Introduction
Polar environments are those with characteristics of low temperature, strong wind, low nutrient content and high ultraviolet radiation. In order to survive in polar environment microorganisms need to develop many kinds of physiological and biochemical adaptations that are usually accompanied by modification of gene regulation and metabolic pathways (Wilson and Brimble 2009). Polar microorganisms can produce a large number of biologically active natural products to deal with extreme environments, and have their unique chemical defense system. Nowadays, researchers have obtained many natural products with good biological activity from polar microorganisms (Pascale et al. 2012). They have many biological activities such as anti-tumor (Kagiyama et al., 2016;Gao et al. 2017), anticancer (Dalsgaard et al. 2004;Ivanova et al. 2007), antibacterial (Giudice et al. 2007), antioxidant (Zhou et al. 2010), antiviral (Cai et al. 2012) and so on. The special characteristics of the polar environment increases the possibility of discovering new active secondary metabolites, showing with various application potential. Alzheimer's disease (AD), is a multi-factor-induced chronic neurodegenerative disease threatened the human health worldwide. Acetyl cholinesterase (AChE) is a key enzyme in biological nerve conduction. Inhibition of AChE can effectively treat AD. Overweight and obesity are defined as abnormal or excessive fat accumulation, and they present a risk to health. Pancreatic lipase (PL) inhibitors play a major role to treat obesity. Thus, it is extremely urgent to discover new AChE and PL inhibitors with novel structures and significant bioactivities.

Results and discussion
All the isolated compounds (1-12) were evaluated for their cytotoxic, antibacterial activities and enzyme inhibitory activities against AChE and PL. These compounds were tested for cytotoxic activities against 143B, C4-2B and 22Rv1 cell lines and antibacterial activities (three Gram-positive bacteria, methicillin-resistant Staphyloccocus aureus (MRSA), Staphyloccocus aureus (ATCC 29213), Enterococcus faecalis (ATCC 29212) and three Gram-negative bacteria, Acinetobacter baumannii (ATCC 19606), Escherichia coli (ATCC 25922), Klebsiella pneumonia (ATCC 13883). However, no compounds showed antibacterial activities at concentration of 50 lg/mL. Compound 5 exhibited moderate in vitro cytotoxic activity against the 143B cell line with IC 50 value of 12.64 ± 0.78 lM (IC 50 1.15 ± 0.08 lM for Cisplatin). Enzyme inhibitory activities against AChE, and PL were also determined according to previously reported method (Pang et al. 2021). Compound 6 showed strong inhibitory activity against AChE with IC 50 value of 0.36 nM (IC 50 18.7 nM for Tacrine), while compounds 6 and 11 showed weak PL enzyme inhibitory activity with IC 50 values of 23.5 and 155.5 lM, respectively.
To investigate the inside interactions between compound 6 and AChE, a preliminary molecular docking study was conducted in AutoDock4.2.6 ( Morris et al. 2009). The highest scoring conformation was obtained as shown in Figure 2. The analysis of the predicted result revealed that compound 6 could tightly bind inside the active pocket of AChE with the calculated binding energy of À11.4 kcal/mol. The benzene ring with three methoxy groups of 6 stretched into the catalytic pocket ( Figure 2A) which consisted of residues His-440, Phe-330, and Ser-200. This catalytic pocket was the same as that tacrine would bind to when exerting its inhibitory effect against AChE (Galdeano et al. 2018). In the active site, four hydrogen bonds between 6 and three residues His-440, Ser-200, and Gly-118 with the lengths of 2.1, 1.7, 2.4, and 2.4 Å, respectively, were observed in 3D molecular binding mode ( Figure 2B). In addition, the pyrone ring of 6 formed a p-p T-shaped interaction with Phe-330. All above interactions contributed to the strong anchoring of 6 to the binding site of AChE, and provided an inside perspective of the action of 6, and a better understanding of the enzyme inhibitory activity.

Fungal material and culture conditions
The fungal strain Penicillium sp. SCSIO 05705 was isolated from a soil sample that was collected nearby the Great Wall station (Chinese Antarctic station, 62 12'59"S, 58 57'52"W). It was identified according to its morphological characteristics and ITS gene sequences (GenBank Accession No. KR824116). A reference culture is deposited in our laboratory at À80 C. The produced strain was stored on MB agar (malt extract 15 g, sea salt 10 g, agar 16 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, Guangzhou, China.

Extraction and isolation
A large-scale fermentation of fungal SCSIO 05705 was incubated at room temperature in 1 L Â 36 conical flasks with solid rice medium (each flask contained 150 g of rice, 4.5 g of artificial sea salt, and 150 mL H 2 O). These flasks were incubated statically at 24 C under a normal day/night cycle. After 55 days, the rice medium was soaked in acetone (400 mL/flask), cut into small pieces and sonicated for 20 min. Then, the acetone was evaporated under reduced pressure to obtain an aqueous solution, which was extracted with EtOAc three times. At the same time, the rice residue was also extracted with EtOAc to give another EtOAc solution. Both of the EtOAc solutions were combined and concentrated under reduced pressure to afford 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 (79 g).

Bioassay for enzyme inhibitory activity
AChE and PL inhibitory activities of compounds 1-14 were assessed according to the spectrophotometric method with slight modification (Pang et al. 2021). Tacrine and Orlistat were used as the positive controls with the IC 50 values of 18.7 nM and 0.67 lM, respectively.

Bioassay for cytotoxic activity
Cytotoxic activities (143B, C4-2B and 22Rv1 cells) were evaluated using the CCK-8 method as described previously (Zheng et al. 2020). Cisplatin was used as the positive control with the IC 50 value of 1.15 ± 0.08 lM.

Bioassay for antibacterial activity
Antibacterial activities were evaluated in 96-well microtiter plates using a modification of the broth microdilution method (Wang et al. 2016).

Conclusion
In this study, a new indole diketopiperazine alkaloid, penilline D (1), was isolated from the Antarctic fungus Penicillium sp. SCSIO 05705, together with eleven known compounds (2-12). Extensive NMR spectroscopic analysis and ECD calculation were used to elucidate the structure of penilline D (1), including its absolute configuration. All the isolated compounds (1-12) were evaluated for their cytotoxic, antibacterial activities and enzyme inhibitory activities against AChE and PL. Among them, compound 5 exhibited moderate in vitro cytotoxic activity against the 143B cell line with IC 50 value of 12.64 ± 0.78 lM. Compound 6 showed strong inhibitory activity against AChE with IC 50 value of 0.36 nM (IC 50 18.7 nM for Tacrine), while compounds 6 and 11 showed weak PL enzyme inhibitory activity. Furthermore, an in silico molecular docking study was also performed between compound 6 and AChE. The results of enzyme inhibitory experiments indicated that compound 6 might be regarded as one of the candidate precursors for the development of AChE enzyme inhibitor.