New Tetramic Acid Derivatives From the Deep-Sea-Derived Fungus Penicillium sp. SCSIO06868 With SARS-CoV-2 Mpro Inhibitory Activity Evaluation

Three new tetramic acid derivatives (1–3) and a new polyketide (4) along with eight known compounds (5–12) were isolated from cultures of the deep-sea-derived fungus Penicillium sp. SCSIO06868. Four new structures were elucidated by analysis of one-dimensional/two-dimensional nuclear magnetic resonance (NMR) data and high-resolution electrospray ionization mass spectrometry. Their absolute configurations were established by X-ray crystallography analysis and comparison of the experimental and reported electronic circular dichroism (ECD) values or specific optical rotation. Compound 3 exhibited potent, selective inhibitory activities against Staphylococcus aureus and methicillin-resistant S. aureus with minimum inhibitory concentration values of both 2.5 μg/ml. Also, compound 3 showed weak antiviral activity against severe acute respiratory syndrome coronavirus 2 main protease, which was responsible for the coronavirus disease 2019 pandemic.


INTRODUCTION
Natural products bearing a tetramic acid structural fragment (pyrrolidine-2,4-dione) are isolated from various terrestrial and marine organisms, such as bacteria, cyanobacteria, fungi, and sponges (Mo et al., 2014;Jiang et al., 2020). Tetramic acids showed a remarkable diversity of bioactivities, including antitumor (Lin et al., 2008;Fan et al., 2020), antiviral (Sun et al., 2015), antibacterial (Nord et al., 2020;Wingen et al., 2020), larvicidal (Mao et al., 2019), and herbicidal (Schrey et al., 2019) activities (Schobert and Schlenk, 2008;Mo et al., 2014;Jiang et al., 2020). Among the different marine sources, marine fungi mainly containing Aspergillus, Penicillium, and Cladosporium species are the dominant sources of the rapidly increasing numbers of tetramic acids (Jiang et al., 2020). With the development of sampling techniques and the possibility to culture organisms from deep-sea even in conventional standard microbiological laboratories, deep-sea-derived fungi have recently received a wide concern as a new area for bioprospecting (Jin et al., 2016;Pang et al., 2020). As part of our ongoing research for bioactive secondary metabolites from deep-sea-derived fungi (Chen et al., 2016;Wang et al., 2016;Pang et al., 2021), the fungus Penicillium sp. SCSIO06868 was studied. Three new tetramic acid derivatives (1-3) and a new polyketide (4) along with eight known compounds (5-12) (Figure 1) were isolated from the deep-sea-derived fungus Penicillium sp. SCSIO06868, which was cultured on a liquid medium. The coronavirus disease 2019  pandemic has left a mark in more than 180 countries, with more than 2.0 billion cases worldwide and over 4.4 million deaths in total (until August 2021). The COVID-19 is an infectious disease caused by a novel strain of coronavirus [severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)] (Zehra et al., 2020;Shamsi et al., 2021). SARS-CoV-2 main proteinase (M Pro ), a key protease of CoV-2, mediates viral replication and transcription. SARS-CoV-2 M Pro has emerged as an attractive target for SARS-CoV-2 drug design and development (Sabbah et al., 2021). All isolated compounds (1-12) were tested for their antiviral activities against SARS-CoV-2 M pro in vitro. Molecular docking research was performed to mimic the interactions between the bioactive compound and SARS-CoV-2 M pro . Herein, we described the isolation, structure elucidation, and bioactivity evaluation of the 12 compounds.

Fungal Material
The strain SCSIO06868 was isolated from the deep-sea sediment collected from the Indian Ocean (94 • 37.377 E; 2 • 59.853 S; depth 4,762 m). The internal transcribed spacer sequences of SCSIO06868 (494 base pairs, GenBank accession no. MZ277624) have 99% sequence identity to that of Penicillium citrinum DUCC5728 (GenBank accession no. 582768). Then, it was designated as a member of Penicillium sp. and named as Penicillium sp. SCSIO06868. The strain SCSIO06868 was stored on methylene blue agar (malt extract 15 g, agar 16 g, sea salt 10 g, water 1 L, pH 7.4-7.8) slants at 4 • C and deposited at Key Laboratory of Tropical Marine Bio-resources and Ecology, Chinese Academy of Sciences.

Fermentation and Extraction
The mass fermentation of this fungus was carried out in 1-L Erlenmeyer flasks. The fungus was inoculated in a liquid medium (2% maltose, 2% mannitol, 1% monosodium glutamate, 1% glucose, 0.3% yeast extract, 0.05% monopotassium phosphate, 0.03% MgSO 4 ·7H 2 O, and 300-ml tap water/flask, 93 flasks, 28 L total) at 25 • C under static condition for 35 days. After 35 days, the fermentation was soaked in ethyl acetate (500 ml/flask), and the mycelia were cut into small pieces and sonicated for 20 min. The ethyl acetate solution was concentrated under reduced pressure to gain a brown crude extract (59.0 g).

Molecular Docking Research
The molecular docking was conducted by AutoDockTools (Version 1.5.6) (Morris et al., 2008). The crystal structure of SARS-CoV-2 main protease (PDB ID: 6LU7) was retrieved from the Protein DataBank 1 (Jin et al., 2020). The structures were generated in ChemBio3D Ultra 14.0 (ChemBioOffice version 14.0), followed by an MM2 calculation to minimize the conformation energy. The original ligand and crystal water were removed before the docking calculation. The hydrogens were added to the structure of 6LU7, and Kollman united partial charges were assigned. A Lamarckian genetic algorithm was applied as a default search algorithm and set the grid box within the size of 46 × 44 × 46 Å, with the spacing of 0.375 Å. During the docking, the default parameters were used if it was not mentioned. The docking pose that had the lowest binding energy was represented as the most favorable binding conformation.

Antibacterial Activity Assay
All compounds (1-12) were tested for antibacterial activities against five pathogenic bacteria using the method of agar filter paper diffusion. Compounds that had inhibition zone were evaluated in 96-well plates using a modification of the broth microdilution method (Pang et al., 2018). Ampicillin and gentamicin were used as a positive control for Gram-positive and Gram-negative bacteria, respectively.

Antiviral Activity Assay
The antiviral activities of all compounds (1-12) against SARS-CoV-2 M pro were evaluated through the method mentioned in the previous report . Hydroxychloroquine showed potent inhibitory activity against SARS-CoV-2 M pro with Ki = 0.36 µm and was used as a positive control.  (Lin et al., 2008;Yoda et al., 2010) showed that they only differed by an absence of the singlet methyl in 1. The planar structure of 1 was further confirmed by its heteronuclear multiple bond correlation (HMBC) correlations (Figure 2) of H-6 to C-4, H 3 -7 to C-5, H 2 -10 to C-8 and C-12, and H 3 -16 to C-8 and C-10. The configuration of a double bond at C-3 was determined as Z based on that the chemical shift of acylamino (δ C 178.0, C-2) was in a lower field than those of normal ones, which caused by the hydrogen bond between the oxygen atom at C-2 and hydroxy at C-8 (Aoki et al., 2000). Thus, the ECD of 1 (Figure 3) displayed a positive Cotton effect at 209 nm ( ε = + 18.39), a negative Cotton effect at 227 nm ( ε = -15.33), and a negative Cotton effect at 281 nm ( ε = -4.38), and the trend of which was consistent with that of 5. Thus, the absolute configuration of C-5 in 1 was determined as S. Mosher's method was tried to confirm the absolute configuration of C-6 but failed. Fortunately, the single crystal of 1 was obtained, and the absolute configuration of 1 was established as 5S, 6R, 9S by analyzing the X-ray crystallographic data (Figure 4). Compound 1 was named as penicillenol G1. The molecular formula of penicillenol G2 (2), which was the same as 1, was established as C 15 H 25 NO 4 by its NMR data ( Table 1) and a protonated molecule at m/z 284.1862 in the HRESIMS. Its NMR data were nearly the same as those of 1, and only the chemical shifts of C-5 (δ C/H 68.1/3.94, brs), C-6 [δ C/H 68.7/4.08 (qd, J = 6.3, 3.5 Hz)], and C-7 [δ C/H 17.0/1.10 (d, J = 6.3 Hz)] in 2 have some small differences. Compound 2 had the same planar structure with 1, which was confirmed by the 1 H-1 H COSY and HMBC spectra (Figure 2). Thereby, the differences mentioned earlier might be caused by their different configurations. The ECD spectrum of 2 showed a negative Cotton effect at 213 nm ( ε = -3.08), a positive Cotton effect at 238 nm ( ε = + 2.5), and a positive Cotton effect at 271 nm ( ε = + 2.89), which exhibited a reverse trend with 1 and 5, but was consistent with penicillenol A2 (6) (Lin et al., 2008;Sengoku et al., 2012). Therefore, the absolute configuration of C-5 in 2 was determined as R. Some synthetic chemists demonstrated that the natural penicillenol A1 (5) (Yoda et al., 2010), penicillenol A2 (6) (Sengoku et al., 2012), and penicillenol C1 (7) (Kempf et al., 2013) were all have the same absolute configurations of 6R and 9S, and the absolute configuration of compound 1 was also determined as 6R and 9S by the X-ray diffraction study. Thus, considering the same biosynthetic pathway (Yin et al., 2019) and comparison of the spectroscopic data with 1, the absolute configurations of C-6 and C-9 in 2 were deduced as 6R, 9S.
Penicillenol H (3) was obtained as a yellow oil. The 13 C NMR data and a protonated molecule at m/z 284.1858 in the HRESIMS of 3 suggested that its molecular formula was C 15 H 25 NO 4 with 4 degrees of unsaturation. The NMR data ( Table 1) of 3 were similar to those of 1, except that the 1-hydroxyethyl group in 1 was displaced by oxygenated methylene (δ C/H 59.3/3.94, qd, J = 12.6, 2.8 Hz, CH 2 -6) and an N-methyl group (δ C/H 26.8/3.03, s, CH 3 -17) was added in 3. The extinction was determined by its 1 H-1 H COSY cross-peak of H 2 -6/H-5 and HMBC correlations of H 2 -6 to C-4 and H 3 -17 to C-2 and C-5. The planar structure of 3 was further established by its 2D NMR (Figure 2). The configuration of a double bond at C-3 was designated as Z by the chemical shift of C-2 (δ C 178.0) (Aoki et al., 2000). The absolute configuration of C-5 and C-9 in 3 were determined as S and R same as compound 1 by their similar ECD spectrum (Figure 3), of which 3 showed a positive Cotton effect at 213 nm ( ε = + 2.87), two negative Cotton effects at 231 nm ( ε = -2.76), and 289 nm ( ε = -1.27), respectively. Compound 3 was named penicillenol H.

Bioassays of Compounds
All isolated compounds (1-12) were tested for their antiviral activities against SARS-CoV-2 M pro in vitro through the method mentioned in the reported literature . Compound 3 showed weak inhibitory activity against M pro enzyme, which was responsible for the COVID-19 pandemic. When treated with 50 µm of 3, the relative enzyme activity of SARS-CoV-2 M pro was 46.64%, and that of the positive control hydroxychloroquine was 5.28% with the same concentration. To better understand the interactions between compounds and SARS-CoV-2 M pro , molecular docking research was performed to mimic the interactions between compound 3 and M pro enzyme of SARS-CoV-2 (PDB ID: 6LU7) by utilizing the AutoDockTools. Molecular docking results demonstrated that compound 3 could interact with the SARS-CoV-2 M pro enzyme at the entrance of the catalytic pocket, with the calculated binding affinities of -4.98 kcal/mol. The 2D binding model for 3 (Figure 5) showed two hydrogen bonds and two intermolecular hydrophobic interactions. Two hydrogen bonds were formed between the carbonyl group at C-4 and Thr-26, as well as between the hydroxyl group at C-6 and Thr-24. The lengths of the two hydrogen bonds were 2.0 and 1.8 Å, respectively. These results suggested that compound 3 could insert into the active site of the enzyme and bind tightly to the catalytic amino acid residues by different types of interactions to inhibit SARS-CoV-2 M pro .
Compounds 1-12 were evaluated their antibacterial activities against five pathogenic bacteria Escherichia coli (ATCC 25922), Enterococcus faecalis (ATCC 29212), Klebsiella pneumonia   Compounds 1, 3, 5, and 6 with 50 µg/disc showed inhibition zones against S. aureus. Compounds 1, 3, and 5 with 50 µg/disc showed an inhibition zone against MRSA (Supplementary Figure 1). Furthermore, their minimum inhibitory concentrations (MICs) were tested, and the results are shown in Table 3. Compound 3 displayed potent inhibitory activities against S. aureus and MRSA with MIC values of both 2.5 µg/ml. Ampicillin was used as a positive control against S. aureus and MRSA with MIC values of 1.56 and 0.39 µg/ml, respectively.

CONCLUSION
In summary, we reported the isolation and identification of three new tetramic acid derivatives (1-3) and a new polyketide (4) along with eight known compounds (5-12) from cultures of the deep-sea-derived fungus Penicillium sp. SCSIO06868. The absolute configurations of new compounds were established by X-ray crystallography analysis and comparison of the experimental and reported ECD value or specific optical rotation. Compound 3 displayed potent inhibitory activities against S. aureus and MRSA with MIC values of both 2.5 µg/ml. Compound 3 showed weak inhibitory activity against M pro enzyme of SARS-CoV-2, which was responsible for the COVID-19 pandemic. Molecular docking was performed to mimic the interactions between compound 3 and SARS-CoV-2 M pro . The molecular docking results indicated that compound 3 could be inserted into the active site of the enzyme and bind tightly to the catalytic amino acid residues by different types of interactions to inhibit SARS-CoV-2 M pro .

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
XP, JW, and YL contributed to conception and design of the study. XP performed the experiments, analyzed the data, and wrote the manuscript. XW performed the SARS-CoV-2 M pro inhibition test. XT did the isolation of the fungus. All authors contributed to manuscript revision, review, and approved the submitted version.