Ferroptosis Inhibitory Compounds from the Deep-Sea-Derived Fungus Penicillium sp. MCCC 3A00126

Two new xanthones (1 and 2) were isolated from the deep-sea-derived fungus Penicillium sp. MCCC 3A00126 along with 34 known compounds (3–36). The structures of the new compounds were established by spectroscopic data. The absolute configuration of 1 was validated by comparison of experimental and calculated ECD spectra. All isolated compounds were evaluated for cytotoxicity and ferroptosis inhibitory activities. Compounds 14 and 15 exerted potent cytotoxicity against CCRF-CEM cells, with IC50 values of 5.5 and 3.5 μM, respectively, whereas 26, 28, 33, and 34 significantly inhibited RSL3-induced ferroptosis, with EC50 values of 11.6, 7.2, 11.8, and 2.2 μM, respectively.


Introduction
The oceans cover over 70% of the world's surface, with 95% of them being deeper than 1000 m. In recent years, nearly half of the new marine natural products (MNPs) have been isolated from marine microorganisms [1][2][3], especially fungi, the most diverse and abundant eukaryotes on Earth, which can be distributed in any currently known extreme environment [4]. As a region rarely explored, the deep sea is characterized by a high pressure, a low/high (such as hydrothermal mouth) temperature, a high salt concentration, the absence of light, oligotrophic conditions, a high halogen content, and so on. To adapt to such extreme environments, deep-sea-derived microorganisms must develop special metabolic mechanisms, giving rise to tremendous secondary metabolites with unique structures and potent bioactivities [5]. For more than half a century, MNPs have been continuously discovered, but those from the deep sea are rare [6,7]. In recent years, with the development of deep-sea sample collection technology, reports of deep-sea MNPs have increased significantly. As an important group of deep-sea microorganisms, fungi can produce a large number of structurally novel and biologically active secondary metabolites, which have attracted extensive attention from researchers. For example, vercytochalasins A and B are two novel, biosynthetically related cytochalasins isolated from Curvularia verruculosa, the endophytic fungus of the deep-sea lobster Shinkaia crosnieri. Vercytochalasin A is the most potent natural product against angiotensin-I-converting enzyme (ACE), with an IC 50 value of 505 nM [8]. Chevalinulins A and B are two indole alkaloids with a rare spiro-[bicyclo [2.2.2]octane-diketopiperazine] skeleton. They both exhibit significant in vivo proangiogenic activity in transgenic zebrafish [9].
Xanthones, also known as 9H-xanthen-9-ones, are a class of yellow compounds bearing a dibenzo-γ-pyrone scaffold. They are widely distributed in plants, lichens, and microorganisms of terrestrial and marine origin, and exhibit diverse biological activities such as antiviral [10], cytotoxic [11], antibacterial [12], antifungal [13], and hypoglycemic [14] activities. The molecular skeleton of xanthones can bind with a variety of targets, so this

Results and Discussion
Compound 1 was obtained as a colorless gum. The molecular formula C 18 H 18 O 9 was determined by the positive HR-ESI-MS (high resolution electrospray mass spectrometry spectrum) at m/z 401.0839 [M + Na] + , suggesting ten degrees of unsaturation. The 1 H ( Figure S1 in the Supplementary Materials) and 13 C ( Figure S2 in the Supplementary Materials) NMR (nuclear magnetic resonance) spectroscopic data (Table 1) Figure 2).

Results and Discussion
Compound 1 was obtained as a colorless gum. The molecular formula C18H18O9 was determined by the positive HR-ESI-MS (high resolution electrospray mass spectrometry spectrum) at m/z 401.0839 [M + Na] + , suggesting ten degrees of unsaturation. The 1 H (Figure S1 in the Supplementary Materials) and 13 C ( Figure S2 in the Supplementary Materials) NMR (nuclear magnetic resonance) spectroscopic data (  3) in the same concentration (c 0.2) and the same solvent (CHCl3), (c 0.2, CHCl3), suggesting they have the same absolute configuration at C-7 and C-8. For the further confirmation, the ECD (electron circular dichroism) spectra were calculated for (7R,8R)-1 (1a) and its enantiomer (7S,8S)-1 (1b) using Yinfo Cloud Computing Platform (https://cloud.yinfotek.com, accessed on: 13, June, 2022). Thirty states of each seven conformers were calculated to generate the ECD curves. As shown in Figure 3, the calculated ECD spectrum of 1a was consistent with that of the experimental one. On the basis of the above evidence, compound 1 was then elucidated as 11-O-acetylaspergillusone B.   3) in the same concentration (c 0.2) and the same solvent (CHCl 3 ), (c 0.2, CHCl 3 ), suggesting they have the same absolute configuration at C-7 and C-8. For the further confirmation, the ECD (electron circular dichroism) spectra were calculated for (7R,8R)-1 (1a) and its enantiomer (7S,8S)-1 (1b) using Yinfo Cloud Computing Platform (https://cloud.yinfotek.com, accessed on 13 June 2022). Thirty states of each seven conformers were calculated to generate the ECD curves. As shown in Figure 3, the calculated ECD spectrum of 1a was consistent with that of the experimental one. On the basis of the above evidence, compound 1 was then elucidated as 11-O-acetylaspergillusone B.  Compound 2 was obtained as a amorphous yellow solid. The molecular formula C17H12O7 was established by its positive HR-ESI-MS spectrum at m/z 351.0482 [M + Na] + . The 1 H and 13 C data of 2 showed the presence of two methoxyls, five methines and ten quaternary carbons, which were closely related to those of huperxanthone A (13) [14], except that the sp 2 quaternary carbon at C-7 (δC 151.1 s) in 13 was changed as an sp 2 methine (δC 123.1 d) in 2. By detailed analysis of its HSQC (heteronuclear single quantum correlation), 1 H-1 H COSY, HMBC, and NOESY (nuclear Overhauser effect) spectroscopic data, compound 2 was then established as 7-dehydroxyhuperxanthone A.
Since the crude extract of Penicillium sp. MCCC 3A00126 showed a potent anti-proliferative effect on CCRF-CEM, all 36 isolates were subjected to cytotoxicity tests on the same acute lymphoblastic leukemia using the CCK-8 assay. As shown in Figure 4, under a concentration of 20 μM, two compounds, 14 and 15, exerted potent activity, with cell survival rates of 6.2% and 7.3%, respectively, while seven compounds, 4, 8, 11, 17, 20, 28,  and 29, showed weak effects, with cell survival rates of 70.2%, 78.5%, 78.8%, 62.3%, 75.8%, 55.3%, and 55.3%, respectively. Interestingly, compounds 14 and 15 possess a difuran ring at C-5 and C-6, which might be the key to the bioactivity.  Ferroptosis is an iron-dependent mode of necroptosis induced by certain small molecules, such as RSL3 (the glutathione peroxidase 4 inhibitor), which is different from apoptosis, necrolysis, and autophagy [50]. Its main characteristics are the generation of ROS (reactive oxygen species), LPO (lipid peroxidation), and iron accumulation. RSL3 acts on specific targets in cells and causes a reduction in antioxidants GSH (glutathione) and GPX4 (glutathione peroxidase 4), resulting in the accumulation of ROS in cells, LPO in cells, and ferroptosis in cells under the synergistic effect of iron [51]. Many tumor cells that are easy to metastasize are prone to ferroptosis, so inducing and inhibiting ferroptosis for pharmacological regulation has great potential in the treatment of certain cancers. Ferroptosis is an iron-dependent mode of necroptosis induced by certain small molecules, such as RSL3 (the glutathione peroxidase 4 inhibitor), which is different from apoptosis, necrolysis, and autophagy [50]. Its main characteristics are the generation of ROS (reactive oxygen species), LPO (lipid peroxidation), and iron accumulation. RSL3 acts on specific targets in cells and causes a reduction in antioxidants GSH (glutathione) and GPX4 (glutathione peroxidase 4), resulting in the accumulation of ROS in cells, LPO in cells, and ferroptosis in cells under the synergistic effect of iron [51]. Many tumor cells that are easy to metastasize are prone to ferroptosis, so inducing and inhibiting ferroptosis for pharmacological regulation has great potential in the treatment of certain cancers.

General Experimental Procedures
The HR-ESI-MS spectra were obtained on a Waters Xevo G2 Q-TOF mass spectrometer equipped with a Spray™ ESI source in both the positive and negative ion mode. NMR

General Experimental Procedures
The HR-ESI-MS spectra were obtained on a Waters Xevo G2 Q-TOF mass spectrometer equipped with a Spray™ ESI source in both the positive and negative ion mode. NMR spectra were recorded in CDCl 3 , CD 3 OD, or DMSO-d 6 on a Bruker Avance III 400 Mz spectrometer at room temperature. Optical rotation was measured by an Anton Paar MCP 100 polarimeter. UV and ECD spectra were acquired on a JASCO J-810 spectropolarimeter. Preparative HPLC (high-performance liquid chromatography) separations for purification were carried out on an Agilent 1260 system with a semi-preparative chromatographic column (COSMOSIL 5C 18 -MS-II, 5PFP, SL-II, 250 mm × 10 mm). Materials for column chromatography (CC) included silica gel, ODS (octadecylsilyl), and Sephadex LH-20.

Biological Material
The deep-sea-derived fungus Penicillium sp. MCCC 3A00126 was isolated from a sediment sample collected from the Eastern Pacific Ocean at a depth of 5246 m by Professor De-Zan Ye of the Third Institute of Oceanography, Ministry of Natural Resources, in September 2003. The 18S rRNA gene sequence alignment (AM18217) showed great similarity (99.88%) to Penicillium sp. PB g (GenBank accession number MK372218.1); therefore, it was identified to be Penicillium sp. It was deposited at the Marine Culture Collection of China (Xiamen, China) with accession number MCCC 3A00126.

Fermentation, Isolation, and Purification
The strains stored at −80 • C were inoculated into a 250 mL conical flask containing 100 mL PDB culture medium to conduct initial activation for three days in a shaking table culture at 28 • C. Then, under the same culture conditions, 1 mL of the fungal liquid was placed in another 1 L conical flask containing 250 mL of PDB medium to conduct secondary activation.
The secondary activated fungal strain was inoculated into 60 Erlenmeyer flasks containing 80 g rice and 120 mL distilled water. The fermentation was kept under static conditions at 25 • C. After 40 days, 400 mL of EtOAc was added to each flask over 12 h. The organic solvent was filtered. The procedure was repeated four times. The organic solvents were combined and concentrated to a small volume. The latter was dissolved in MeOH and extracted by PE (petroleum ether) three times. The MeOH layer was concentrated to provide a crude extract (26.3 g), which was subjected to MPLC (medium-pressure liquid chromatography) over silica gel using the CH 2 Cl 2 /MeOH gradient (100% → 70%) to obtain four fractions (Fr

ECD Calculation
ECD calculations were performed using Yinfo Cloud Computing Platform, a user-friendly and versatile web server for biomedicinal, material, and statistical research (https://cloud. yinfotek.com, accessed on 13 June 2022). The conformational analysis of compound 1 was carried out using the MMFF94 force field at an energy cutoff of 7.0 kcal/mol and an RSMD threshold of 0.5 Å. A total of thirteen conformations were obtained from the conformational analysis, and seven of which, accounting for more than 1%, were selected for further screening. The seven conformers were relocated and confirmed at the PM6, HF/6-31G(d), and B3LYP/6-1G(d) level to obtain three dominant conformers. Further, the calculation of the theoretical ECD spectra at the B3LYP/6-311G(d, p) level was conducted in MeOH. The final spectrum was obtained by averaging each conformer using the Boltzmann distribution.

Cytotoxic Experiment
CCRF-CEM cells (CL-0058), kindly provided by Procell Life Science & Technology Co., Ltd. (Wuhan, China), were cultured in RIPM-1640 at 37 • C in a humidified atmosphere containing 5% CO 2 with 10% inactive fetal bovine serum, 2 mM L-glutamine, 100 IU penicillin, and 100 mg/mL streptomycin. The cytotoxicity experiment was conducted using the CCK-8 (Cell Counting Kit-8) assay. Briefly, 4000 cells were seeded on a 96-well plate. After 24 h, different concentrations of the tested compounds were added, and the incubation continued for 48 h. The CCK-8 assay was performed with MD Spectra Max Paradigm.

Ferroptosis Inhibitory Assay
As previously reported [54], 10,000 CCRF-CEM cells were seeded on a 96-well plate for 24 h. Then, ferrostatin-1 (1 µM, as the positive control) and different concentrations of the tested compounds, ranging from 1 µM to 20 µM, were added for first-round screening. After 0.5 h, RSL3 (2 µM) was added to trigger ferroptosis. Four hours later, cellular ATP was detected using the Cell Titer Glo Luminescent Cell Viability assay kit (G7570, Promega, Madison, WI, USA) according to the manufacturer's instructions. Then, the EC 50 values were determined as the indicated concentration.

DPPH Assay
According to a reported procedure [55], the stable radical DPPH (2,2-diphenyl-1picrylhydrazyl) was dissolved in MeOH to a final working concentration of 100 µM. Then, 1 µL of the indicated compounds dissolved in DMSO (10 mM) was added to a final concentration of 100 µM, inverted several times, and allowed to incubate at room temperature for 30 minutes. Samples were then aliquoted to a 96-well microplate and absorbance at 517 nm was recorded using Spectra Max Paradigm (Molecular Devices, San Jose, CA, USA). The relative DPPH normalized to the background (MeOH only) showed the mean ± SD, and the experiments were triplicated.

Conflicts of Interest:
The authors declare no conflict of interest.