Rare Carbon-Bridged Citrinin Dimers from the Starfish-Derived Symbiotic Fungus Penicillium sp. GGF16-1-2

Four novel, rare carbon-bridged citrinin dimers, namely dicitrinones G–J (1–4), and five known analogs (5–9) were isolated from the starfish-derived fungus Penicillium sp. GGF 16-1-2. Their structures were elucidated by extensive spectroscopic analysis and quantum chemical calculations. Compounds 1–9 exhibited strong antifungal activities against Colletotrichum gloeosporioides with LD50 values from 0.61 μg/mL to 16.14 μg/mL. Meanwhile, all compounds were evaluated for their cytotoxic activities against human pancreatic cancer BXPC-3 and PANC-1 cell lines; as a result, compound 1 showed more significant cytotoxicities than the positive control against both cell lines. In addition, based on the analyses of the protein-protein interaction (PPI) network and Western blot, 1 could induce apoptosis by activating caspase 3 proteins (CASP3).


Introduction
Dimeric natural products are a group of structurally diverse, biologically active, and biosynthetically complex metabolites. Among them, the methylene-bridged ones are a small but unique class that tend to be formed through the dimerization of two monomeric units with a methylene [1]. To date, approximately 131 natural dimers with a methylene linker were reported, including dimeric xanthones, dimeric steroids, and dimeric bioflavonoids [2,3]. Citrinin dimers are formed by the reaction of two citrinins or its analogues, including Diels-Alder-type dimers, 7, 7 carbon-bridged citrinin dimers, and others. Among them, only seven 7, 7 carbon-bridged-type dimers have been reported [4][5][6]. As is known to us, citrinin was one of the well-known mycotoxins [7], while citrinin dimers showed significant bioactivities compared to citrinin monomer derivatives due to their special carbon bridged skeleton, such as anti-fungi, cytotoxicity, and enzyme inhibitory activities [8][9][10].
Mar. Drugs 2022, 20, x FOR PEER REVIEW 2 of 13 studied their antifungal bioactivities against Colletotrichum gloeosporioides and antitumor activities against human pancreatic cancer cells BXPC-3 and PANC-1. The mechanism of cytotoxic activities was proposed via a protein-protein interaction network and Western blot.

Structural Identification of New Compounds
Dicitrinone G (1) was isolated as orange-yellow amorphous powder with a molecular formula of C 25 Figures S4 and S5).
Meanwhile, the NOESY correlation of H-4 and H 3 -9 suggested that H-4 and H 3 -9 were in the β-orientation, while the H-3 and H 3 -10 were in the α-orientation in fragment B ( Figure 3). Compound 1 represents a rare carbon skeleton because of the two citrinin analogues decarboxylates connected through a unique carbon-bridging center. Biosynthetically, this rare skeleton is proposed to originate from a polyketide pathway [24] and the absolute configurations of C-3, C-4, C-3 , and C-4 were established as 3R, 4S, 3 R, 4 S [14,18,25]. To verify the absolute configurations of 1, the quantum chemical ECD calculation was performed. By comparing the predicted ECD curves and the experimental curves, the absolute configuration of C-3, C-4, C-3 , and C-4 of 1 was unambiguously assigned as 3R, 4S, 3 R, 4 S (Figure 4).  Figure S9). established the planar structure of 1 by linking the above two fragments via C-1″ ( Figure  2). Since 1 has two stereoclusters separated by two six-membered rings joined by a methylene group, the relative configuration of each stereocluster was addressed independently. The NOESY correlation of H-4 and H3-9 indicated these protons were cofacial and situated in the α-orientation randomly, and the NOESY correlation of H-3 and H3-10 suggested that H-3 and H3-10 were β-oriented in fragment A (Supplementary Materials Figure S10). Meanwhile, the NOESY correlation of H-4′ and H3-9′ suggested that H-4′ and H3-9′ were in the β-orientation, while the H-3′ and H3-10′ were in the α-orientation in fragment B (Figure 3). Compound 1 represents a rare carbon skeleton because of the two citrinin analogues decarboxylates connected through a unique carbon-bridging center. Biosynthetically, this rare skeleton is proposed to originate from a polyketide pathway [24] and the absolute configurations of C-3, C-4, C-3′, and C-4′ were established as 3R, 4S, 3′R, 4′S [14,18,25]. To verify the absolute configurations of 1, the quantum chemical ECD calculation was performed. By comparing the predicted ECD curves and the experimental curves, the absolute configuration of C-3, C-4, C-3′, and C-4′ of 1 was unambiguously assigned as 3R, 4S, 3′R, 4′S ( Figure 4).   Since 1 has two stereoclusters separated by two six-membered rings joined by a methylene group, the relative configuration of each stereocluster was addressed inde pendently. The NOESY correlation of H-4 and H3-9 indicated these protons were cofacia and situated in the α-orientation randomly, and the NOESY correlation of H-3 and H3-10 suggested that H-3 and H3-10 were β-oriented in fragment A (Supplementary Material Figure S10). Meanwhile, the NOESY correlation of H-4′ and H3-9′ suggested that H-4 and H3-9′ were in the β-orientation, while the H-3′ and H3-10′ were in the α-orientation in fragment B (Figure 3). Compound 1 represents a rare carbon skeleton because of the two citrinin analogues decarboxylates connected through a unique carbon-bridging cen ter. Biosynthetically, this rare skeleton is proposed to originate from a polyketide path way [24] and the absolute configurations of C-3, C-4, C-3′, and C-4′ were established a 3R, 4S, 3′R, 4′S [14,18,25]. To verify the absolute configurations of 1, the quantum chemi cal ECD calculation was performed. By comparing the predicted ECD curves and the experimental curves, the absolute configuration of C-3, C-4, C-3′, and C-4′ of 1 was un ambiguously assigned as 3R, 4S, 3′R, 4′S (Figure 4).   Table 1), indicating the same type of fragment. Compared with 1, 2 showed an additional methyl signal [δ H 1.55, d, (7.4) and δ C 16.4 (CH 3 )] and methine signal [δ H 4.89, q (7.5), and δ C 24.1 (CH)], while the methylene signal [δ H 3.62, q (8.6) and δ C 17.2 (CH 2 )] in 1 was absent in 2, indicating that 2 is a C-1 -CH 3 derivative of 1 (Supplementary Materials Figures S14 and S15). The 1 H-1 H COSY correlation information between H-1 (δ H 4.89, q, 7.5)/H-2 (δ H 1.55, d, 7,4) and the HMBC correlations from H 3 -2 (δ H 1.55, d, 7.4) to C-1 (δ C 24.1, CH), C-7 (δ C 115.7, C) and C-7 (δ C 117.1, C) were further confirmed with the above proposal ( Figure 2).   Similarly, the relative configuration of 2 was deduced based on the NOESY correlations and the absolute configuration was established as 3R, 4S, 1 R, 3 R, 4 S according to biological pathways [24] and the quantum chemical ECD calculation. Dicitrinone I (3) was isolated as orange-yellow colloidal with a molecular formula of C 25 (Table 1) spectra of 3 with those of 1 suggested 3 was highly similar to 1. The major differences between them were the appearance of an oxygenated methylene signal [δ H 4.49 (s) and δ C 59.0 (CH 2 )] in 3 instead of a carbonyl carbon signal [δ C 168.8 (C)] in 1, suggesting that 3 is a hydrogenated analogue of 1. The observed HMBC correlations from H 2 -1 (δ H 4.49, s) to C-8a (δ C 114.6, C), C-8 (δ C 147.9, C), C-4a (δ C 135.1, C), and C-3 (δ C 73.3, CH) were further confirmed based on the above assumption.
Similar to 1, based on the NOESY correlations, the relative configuration of 3 was deduced and based on the subsequent analyses of the biological pathways [24] and the quantum chemical ECD calculation, the absolute configuration of 3 was established as 3R, 4S, 3 R, 4 S.

Evaluation of Antifungal Activity
Colletotrichum gloeosporioides, an important phytopathogenic fungus, mainly infects tropical fruits and results in serious anthracnose [26]. Compounds 1-9 were tested by mycelial growth rate assay against Colletotrichum gloeosporioides [27]. The results ( Table 2) showed that 6 had the strongest antifungal activities against Colletotrichum gloeosporioides with LC 50 values of 0.61 µg/mL. According to their structural characteristics, citrinin monomers and methylene bridges may be important to the antifungal activities. Their bioactivities decreased when the methylene bridge was replaced by an alkane, or the length of the alkane bridge was increased. Meanwhile, if citrinin was oxidized or reduced, its antifungal activities would be weakened.

Cytotoxic Assays
As there is no clinically effective drug for pancreatic cancer [28], we selected Doxorubicin hydrochloride as the positive control for the cytotoxic activity test. Based on the cytotoxicity assay [29] (Table 3), 1 was more significantly cytotoxic against human BXPC-3 cell lines than the Doxorubicin hydrochloride and similar results were obtained against human PANC-1 cell lines with the positive control.
To explore the possible mechanism of the cytotoxic activity of 1, the top 105 potential mechanism genes for 1 have been predicted with the Swiss Target Prediction database [30]; genes related to pancreatic cancer were selected from the GeneCards database to construct the receptor database and 94 overlapping genes were obtained from the Venn diagram. A protein-protein interaction (PPI) network was established by the STRING 11.5 database [31] and Cytoscape 3.9.0 software to screen the critical targets and the size and the color of the symbols represented the degree scores in the network analysis [32]. The analysis results of the PPI indicated that CASP3 has the most degree scores, which means CASP3 could be the essential protein for 1 (Figure 5 A,B). We verified it via a Western blot experiment in vitro and found that 1 could affect CASP3 expression ( Figure 5C), which reportedly plays a crucial role in the cell apoptosis pathway [33][34][35]. Therefore, we hypothesized that 1 might promote BXPC-3 apoptosis by affecting the activation of CASP3. Table 3. Cytotoxic activities of 1-9 in BXPC-3 and PANC-1 cell lines (IC 50 , µM). which means CASP3 could be the essential protein for 1 (Figure 5 A, B). We verified it via a Western blot experiment in vitro and found that 1 could affect CASP3 expression ( Figure 5C), which reportedly plays a crucial role in the cell apoptosis pathway [33][34][35]. Therefore, we hypothesized that 1 might promote BXPC−3 apoptosis by affecting the activation of CASP3.

General Experimental Procedures
Details of the instrumentations and materials used in this work are included in the Supporting Information.

General Experimental Procedures
Details of the instrumentations and materials used in this work are included in the Supplementary Materials.

Fungal Materials, Extraction, and Fermentation
Penicillium sp. GGF 16-1-2 fungus was isolated from starfish in the South China Sea [36]. The strain was stored in the Laboratory of Marine Natural Medicine, School of Pharmaceutical Sciences, Guangzhou University of Chinese Medicine (No. GGF16-1-2).
The fungus Penicillium sp. GGF 16-1-2 was cultured under static conditions at 28 • C in 1 L Erlenmeyer flasks containing 400 mL of the culture medium comprising 10 g monosodium glutamate, 40 g maltose, 13 g yeast extract, 3 g magnesium sulfate heptahydrate, 5 g monopotassium phosphate, 5 g tryptophan, 1 L pure water, and 50 g sorbitol (in seawater). After 60 days of cultivation, 30 L of whole broth was filtered through cheesecloth to separate the supernatant from the mycelia. The former was extracted three times with EtOAc. The culture was extracted thrice with EtOAc and the pooled organic solvent was evaporated to dryness under vacuum to afford a crude extract (111.9 g).
Soybean culture medium: 50 g (≥24 mesh) soybean grains, 33% sea salt, 85 mL pure water, pH natural. A 5.0 mL seed solution was inoculated into soybean culture medium (50 g/1 L/bottle) and a total of 10 L was cultured. The medium was placed in a room at 28 • C for 45 days. The ethyl acetate extract was soaked with ethyl acetate (500 mL ethyl acetate/bottle, 24 h/time, 3-5 times). The ethyl acetate extract was condensed under reduced pressure to yield 5.8 g of the EtOAc residue.

Isolation
The EtOAc soluble fraction (111.9 g) was subjected to a silica gel column chromatography (Si CC,

Antifungal Activity Assay
Initial evaluations of the antifungal activity of the purified compounds were conducted against Colletotrichum gloeosporioides by mycelial growth rate assay. Compound solutions with different concentrations were prepared (three replicates for each concentration) and poured into petri dishes for later use. A PDA medium plate with sterile water was used as a control. The cultured pathogen cakes were taken with a sterile perforator and inoculated into the center of the PDA medium plate. The pathogen cakes were placed in a constant temperature incubator at 28 • C for 3 days. The positive control was carbendazim. The colony diameter was measured by the cross-bonded method and the inhibition rate of mycelium growth was calculated.

Cytotoxic Assays
The toxicity vitalities of 1-9 and doxorubicin hydrochloride were examined by the MTT assays. A 100 µL cell suspension in the culture medium was added into a 96-well plate with a seeding density of 7000 cells per well. The plate was incubated at 37 • C in 5% CO 2 for 12 h. Then the medium was replaced with freshly prepared growth media containing 1-9 at different concentrations of 0 µM, 1 µM, 2 µM, 4 µM, 8 µM, 16 µM, 32 µM, 64 µM, and 128 µM. After 24 h of incubation, 20 µL of 5 mg/mL MTT solution was then added to each well. After 4 h, the MTT medium was removed and 200 µL DMSO was added to each well. After incubating for 10 min, the absorbance at 570 nm was determined with a plate reader.