Cytoprotective Polyketides from Sponge-Derived Fungus Lopadostoma pouzarii

The new polyketides lopouzanones A and B, as well as the new 1-O-acetyl and 2-O-acetyl derivatives of dendrodochol B, were isolated from the sponge-derived marine fungus Lopadostoma pouzarii strain 168CLC-57.3. Moreover, six known polyketides, gliorosein, balticolid, dendrodolide G, dihydroisocoumarine, (–)-5-methylmellein, and dendrodochol B, were identified. The structures of the isolated compounds were determined by a combination of NMR and ESIMS techniques. The absolute configurations of the lopouzanones A and B were determined using the Mosher’s method. The cytotoxicity of the isolated compounds against human prostate cancer cells PC-3 and normal rat cardiomyocytes H9c2 was investigated. Gliorosein showed weak DPPH radical-scavenging activity and in vitro cardioprotective effects toward rotenone toxicity and CoCl2-mimic hypoxia.


Introduction
The marine fungi associated with marine invertebrates, especially sponges, along with the fungi of marine sediments and marine plants, are the main producers of new biologically active compounds [1].
From 1998 to 2017, fungi were found to be an important component of sponge-derived microbes-up to 73%, while actinomycetes and bacteria account for 16% and 11% of spongederived microbes [2]. Additionally, nearly a fifth of all new compounds from marine fungi were isolated from sponge-associated ones from 2010 to 2020 [1,[3][4][5].
The association between sponges and fungi is very interesting for study because the level of relationship between these two groups of organisms is not entirely clear [6]. Perhaps the sponge and its associated fungi are linked by symbiosis, or there is a different type of interaction. Sponges have been studied for a long time as a source of cytotoxic substances [7]. However, several cases are known where the substances isolated from a sponge were then found in the microbials isolated from this sponge. For instance, antimalarial alkaloid manzamine A, isolated from a marine sponge Acanthostrongylophora aff. ingens [8], was later isolated from its symbiont Micromonospora sp. [9]. 1,2-Bis(1H-indol-3-yl) ethane-1,2-dione, an indole alkaloid from the marine sponge Smenospongia sp. [10], was later isolated from marine fungus Penicillium vinaceum, associated with marine sponge Hyrtios erectus [11].
The Lopadostoma pouzarii fungus was first isolated from the Fraxini excelsioris tree and identified as a new species in the Lopadostoma genus (Xylariaceae, Xylariales) [12]. Later two cultures of this fungus were sequenced and found not to belong to Lopadostoma, but they perhaps belong to another genus in the family Xylariaceae, probably Whalleya [13]. Nonetheless, both the Lopadostoma and the Whalleya genera have apparently not been studied much in terms of their secondary metabolites [14]. Only antimicrobial azaphilone pigments sassafrins A-D were isolated from another Lopadostomaceae fungus Creosphaeria sassafras [15]. Other Xylariales fungi are more studied and produced most of the various polyketides [14].
In the continuation of our investigation of the biodiversity and secondary metabolites of Vietnamese marine fungi [16,17], the Lopadostoma pouzarii strain 168CLC-57.3 was isolated from an unidentified marine sponge and cultured for low-molecular weight metabolite isolation.
Herein, we report the isolation, structure elucidation, and biological activity of ten compounds, including four new ones (Figure 1) from the marine sponge-derived fungus L. pouzarii.
The molecular formula of compound 2 was determined as C 10 H 16 O 5 (same as 1), based on the HRESIMS data (m/z 239.0889 [M+Na] + ) corresponding with the 13 C NMR data. An analysis of the NMR data of 2 (Table 1, Figures S12-S16 from Supplementary Materials) and a comparison them with those for lopouzanone A (1) showed the chemical shift differences, especially in the chiral part of the molecule. On the other hand, the HMBC correlations of 2 ( Figure S15 from Supplementary Materials) were in complete agreement with those for 1, which proved the same planar structure.  The relative configurations of 2 were assigned based on the ROESY correlations ( Figure 3 and Figure S17 from Supplementary Materials) between H-4 (δ H 4.25), H-6 (δ H 2.32), and H 3 -7 (δ H 1.19). The absolute configuration of 2 was established by the modified Mosher's method [18]. The esterification of 2 with (S)-and (R)-MTPA chloride occurred at the C-4 hydroxy group to yield the (R)-and (S)-MTPA esters 2a and 2b, respectively. The observed chemical shift differences ∆δ(δ S -δ R ) ( Figure 4) indicated the 4R configuration, and therefore, the absolute configurations of 2 were established as 4R,5R,6S. Compound 2 was named lopouzanone B.

Biological Activity of Isolated Compounds
Compounds 6 and 7 were isolated in an insufficient amount; so, their biological activity was not estimated. The radical-scavenging activity of compounds 1-5, 8, and 9 was determined by DPPH assay (Table 3). Gliorosein (3) showed a statistically significant DPPH-radical scavenging activity and decreased the amount of DPPH radical by 13.9% at a concentration of 100 µM. 102.7 ± 1.8 >100 >100 Ascorbic acid 10.5 ± 3.2 * The differences between this and the control are significant with p ≤ 0.05. 1 The effect of compounds at a concentration of 100 µM.
The cytotoxic activity of compounds 1-5, 8, and 9 toward human prostate cancer PC-3 cells and the rat normal cardiomyocytes H9c2 line is presented in Table 3. The investigated compounds showed a moderate or weak cytotoxic activity. All the compounds at a concentration of 100 µM showed the same cytotoxicity for the rat normal cardiomyocytes H9c2, and none of them decreased H9c2 cell viability by more than by 42%. At a concentration of 10 µM, all the compounds decreased the H9c2 viability by 3-15%. The compounds 1, 2, 5, 8, and 9 showed a weak cytotoxic activity toward the human prostate cancer PC-3 cells. They decreased the PC-3 cell viability by only 27-32% at a concentration of 100 µM; so, its concentrations of 50% inhibition were not calculated. The compounds 3 and 4 were more toxic for PC-3 cells, with the IC 50 of 58.9 µM and 38.9 µM, respectively.
The cytotoxic activity of compounds 1-5 at different concentrations is presented in Figure 5. The cardioprotective effects of the isolated compounds against rotenone and cobalt chloride (II) were investigated. The influence of compound 3 at a nontoxic concentration of 10 µM on the viability of rotenone-and CoCl 2 -treated cardiomyocytes H9c2 is presented in Figure 6. The treatment of H9c2 cells with CoCl 2 decreased the viability of H9c2 cells by 54.1%. Compound 3 at 10 µM increased the viability of CoCl 2 -treated H9c2 cells by 23.5%.

Discussion
So, ten polyketides were isolated from the Lopadostoma pouzarii fungus, which was isolated from the marine environment for the first time. Known gliorosein (3) was reported earlier only from terrestrial fungi Gliocladium spp. [25].
The biosynthesis of isolated new compounds was suggested (Figure 7). We supposed that the biogenesis of lopouzanones A and B was very probably related with gliorosein (3). As was investigated earlier [26], gliorosein originates from tetraketyde 5-methylorsellinic acid. A hydroxylation of the C-5 atom of gliorosein (3) gives lopouzanone B (2), and the reduction and hydroxylation of the direct precursor of gliorosein gives lopouzanone A (1). Dendrodochol B (8) and its derivatives 9 and 10 obviously originate from the heptaketyde precursor that cyclizes to acyl orsellinic acid, the main structural features of which are in structures of 8-10. The consistent reduction, hydroxylation, side chain dihydroxylation, and stereoselective reduction of the aromatic ring yield dendrodochol B and its acetylated derivatives. This is the first report about the selective cytotoxic activity of gliorosein (3) and balticolide (4) against the human prostate cancer PC-3 cells. Earlier, gliorosein was reported to be an antimicrobial compound [27], and selective cytotoxic activity against the HCT116 cell line was reported for the closely related macrolides balticolide [21] and dendrodolide E [22].
At a nontoxic concentration, gliorosein (3) showed cardioprotective activity against the rotenone-and CoCl 2 -induced damage of the cardiomyocytes H9c2.
The treatment of cultured cells with CoCl 2 is well known and widely used in the in vitro model of hypoxia [28]. Hypoxia causes various pathological processes in the cells, including oxidative stress via the blocking of the mitochondrial respiratory chain, which results in an increase in the reactive oxygen species levels into cells [29]. CoCl 2 also induces oxidative stress into H9c2 cardiomyocytes via ROS formation [30]. The natural isoflavonoid rotenone used for the in vitro and in vivo modeling of Parkinson's disease inhibits the mitochondrial complex I, resulting in oxidative stress and cell damage [31]. The inhibition of mitochondrial complex I in H9c2 cardiomyocytes also alters their pro/antioxidant system balance and cell viability [32].
The antioxidant defense against oxidative stress in fungi as well as other eukaryotes is a network of enzymatic superoxide dismutase, catalases, and the thioredoxin system. The fungal response to oxidative stress is therefore complex and necessitates the collaborative involvement of cognate regulators such as the HOG pathway, Yap1, and Skn7 [33]. Nonenzymatic radical-scavenging secondary metabolites have overlapping roles in intercepting ROS and in turning over the cellular buffer of reductants. Polyketides are one of these radical-scavenging compounds. Gliorosein (3) showed a weak radical-scavenging activity in a cell-free assay and can protect H9c2 cells from rotenone-and hypoxia-induced death via ROS scavenging. The anti-radical effect of gliorosein (3) in the DPPH test was not very high, but it was statistically significant. This effect is difficult to correlate directly with its intracellular action on ROS since the difference in the concentration of free radicals in the cell and in the DPPH test can be significant. Earlier, we observed how other orsellinic acid derivatives showed a weak activity in the DPPH test and were effective in a cellular model of Parkinson's disease and were also associated with oxidative stress and ROS [34]. Nevertheless, the cardioprotective properties of gliorosein (3) in hypoxia conditions need future investigations.

Fungal Material and Fermentation
The fungal strain 168CLC-57.3 was isolated from an unidentified sponge sample collected at Cu Lao Cham Island, Quang Nam, Vietnam. The fungus was identified as Lopadostoma pouzarii by gene sequence analysis of the ITS region. According to BLAST analysis, this fungus had the highest similarity to the L. pouzarii strain LPO (89.88%, GenBank accession number KC774601). The strain is currently stored in the NITRA Collection of Marine Microorganisms of the Nhatrang Institute of Technology Research and Application, Vietnam, under code 168CLC-57.3 (GenBank accession number MH752441).

DPPH Scavenging Assay
The compounds were dissolved in DMSO, and the solutions (120 µL) were dispensed into wells of a 96-well microplate. In all, 30 µL of the DPPH (Sigma-Aldrich, Steinheim, Germany) solution in MeOH (1.5 × 10 −4 M) was added to each well. The concentrations of the compounds in mixture were 10 and 100 µM. Pure DMSO was used as a control. The ascorbic acid was used as a positive control. The mixture was shaken and left to stand for 30 min, and the absorbance of the resulting solution was measured at 520 nm with a microplate reader MultiscanFC (ThermoLabsystems Inc., Beverly, MA, USA). The radical scavenging activity of all compounds at 100 µM was presented as a percent of the DMSO data.

Cell Viability Assay
The PC-3 and H9c2 cells were seeded at concentrations of 5 × 10 3 cell/well and 3 × 10 3 cell/well, respectively, and the experiments were started after 24 h. The compounds at concentrations up to 100 µM were added into the wells for 48 h, and the viability of the cells was measured by an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, which was performed according to the manufacturer's instructions (Sigma-Aldrich, Munich, Germany). All compounds were diisolved with DMSO so that the final concentration of DMSO in the cell culture was not more than 1%. Moreover, DMSO was used as a control.
The results were presented as a percent of the control data, and the concentration of the cell viability inhibition on 50% (IC 50 ) was calculated.

Cardioprotective Activity of Compounds in CoCl 2 -Mimic Hypoxia
The H9c2 cells were treated with a dH 2 O-solution of CoCl 2 at a concentration of 500 µM for 2 h. Then, compounds at a concentration of 10 µM were added for 46 h. The viability of the H9c2 cells was measured by an MTT assay.

Cardioprotective Activity of Compounds against Rotenone-Induced Toxicity
The H9c2 cells were treated with rotenone (0.1% DMSO solution) at a concentration of 10 µM for 1 h. Then, compounds at a concentration of 10 µM were added for 23 h. The viability of the H9c2 cells was measured by an MTT assay.

Statistical Data Evaluation
All the data were obtained in three independent replicates, and the calculated values were expressed as mean ± standard error mean (SEM). A Student's t-test was performed using SigmaPlot 14.0 (Systat Software Inc., San Jose, CA, USA) to determine the statistical significance. The differences were considered statistically significant at p < 0.05.