Antiviral 4-Hydroxypleurogrisein and Antimicrobial Pleurotin Derivatives from Cultures of the Nematophagous Basidiomycete Hohenbuehelia grisea

4-Hydroxypleurogrisein, a congener of the anticancer-lead compound pleurotin, as well as six further derivatives were isolated from the basidiomycete Hohenbuehelia grisea, strain MFLUCC 12-0451. The structures were elucidated utilizing high resolution electron spray ionization mass spectrometry (HRESIMS) and 1D and 2D nuclear magnetic resonance (NMR) spectral data and evaluated for their biological activities; for leucopleurotin, we provide Xray data. While most congeners showed moderate antimicrobial and cytotoxic activity, 4-hydroxypleurogrisein emerged as an inhibitor of hepatitis C virus infectivity in mammalian liver cells.


Introduction
Fungi are known as talented producers of secondary metabolites [1]. With previously mainly ascomycetes studied for their potential to produce antibiotic agents, in recent years, basidiomycetes have become the center of attention in the search for new bioactive secondary metabolites [2] and the last group of antibiotics entering the market were the basidiomycete-derived pleuromutilins [3]. Another promising basidiomycete metabolite is the anti-cancer lead compound pleurotin (1), first isolated in 1947 from "Pleurotus griseus Peck" [4], which is now classified as Hohenbuehelia grisea (Peck) Singer. Pleurotin (1; Figure 1) and its derivatives leucopleurotin (2) and dihydropleurotinic acid (3) were shown to have activity against Gram-positive bacteria [4,5] and pathogenic fungi [6], as well as to exhibit anticancerogenic effects [5,7]. Moreover, the total synthesis of (±)-pleurotin (1) [8] and its production in multi-gram scale by fermentation [9] have already been accomplished. In recent years, pleurotin has become the center of attention as a potential new anti-cancer lead drug for its highly effective inhibition of the thioredoxin (Trx)-thioredoxin reductase (TrxR) system [10], a favorable target in the treatment of cancer as well as mercury intoxication [11].
In a concurrent study, searching for promising new pleurotin derivatives from submerged cultures of H. grisea strain MFLUCC 12-0451, three cysteine-derived congeners of pleurotin, thiopleurotinic acids A and B, and pleurothiazole have been found, indicating a potential glutathione detoxification of basidiomycetes [12]. This discovery prompted us to investigate extracts of the strain more closely. Scale up of production and extensive, challenging chromatography procedures of the resulting crude extracts led to the isolation of seven pleurotin derivatives (Figure 2), of which three (5, 9, and 11) are entirely unprecedented. The other four metabolites (6-8 and 10) had been mentioned in the past as potential biosynthesis precursors of pleurotin during the course of pioneer studies on the biosynthesis of pleurotin in the group of Arigoni (ETH Zurich), summarized in Capaul (1992) [13]. However, the corresponding PhD theses contain no spectral data of these compounds, and they may vary in stereochemistry. The present paper is dedicated to the description of their isolation, structure elucidation and biological characterization.
Molecules 2018, 23, x FOR PEER REVIEW 2 of 12 for its highly effective inhibition of the thioredoxin (Trx)-thioredoxin reductase (TrxR) system [10], a favorable target in the treatment of cancer as well as mercury intoxication [11]. In a concurrent study, searching for promising new pleurotin derivatives from submerged cultures of H. grisea strain MFLUCC 12-0451, three cysteine-derived congeners of pleurotin, thiopleurotinic acids A and B, and pleurothiazole have been found, indicating a potential glutathione detoxification of basidiomycetes [12]. This discovery prompted us to investigate extracts of the strain more closely. Scale up of production and extensive, challenging chromatography procedures of the resulting crude extracts led to the isolation of seven pleurotin derivatives ( Figure  2), of which three (5, 9, and 11) are entirely unprecedented. The other four metabolites (6-8 and 10) had been mentioned in the past as potential biosynthesis precursors of pleurotin during the course of pioneer studies on the biosynthesis of pleurotin in the group of Arigoni (ETH Zurich), summarized in Capaul (1992) [13]. However, the corresponding PhD theses contain no spectral data of these compounds, and they may vary in stereochemistry. The present paper is dedicated to the description of their isolation, structure elucidation and biological characterization.  (2), dihydropleurotinic acid (3) [5], and pleurogrisein (4) [13].

Results and Discussion
As reported earlier, the producing strain MFLUCC 12-0451 was identified as Hohenbuehelia grisea (Peck) Singer [12]. H. grisea, more commonly known under its former name of the asexual morph Nematoctonus robustus, has been reported to produce pleurotin (1), dihydropleurotinic acid

Results and Discussion
As reported earlier, the producing strain MFLUCC 12-0451 was identified as Hohenbuehelia grisea (Peck) Singer [12]. H. grisea, more commonly known under its former name of the asexual morph Nematoctonus robustus, has been reported to produce pleurotin (1), dihydropleurotinic acid (2), and leucopleurotin (3) [5]. The group of Arigoni (ETH Zurich) has in the past extensively studied the biosynthesis of pleurotin and in the course of their research proposed several structures [13], which are also being reported with full NMR spectral data in this study for the first time.
3-Hydroxy-dihydropleurotinic acid (5) was isolated as an off-white to pale yellow powder with a molecular formula of C 21 H 24 O 6 , retrieved from a molecular ion cluster [M + H] + at m/z 373.1661, indicating ten degrees of saturation. The correlation of 1 H and 13 C-NMR data ( Table 1) in combination with HMBC correlations (Figure 3) led to the establishment of dihydropleurotinic acid as the underlying core structure [5,12], with the novelty of compound 5 being a hydroxyl functionality at C-3 (δ C 82.8), which leads to the absolute configuration of 3S,4S,5S,8R,9S,10R,15S.
Highly similar to 5 is compound 6, 14-hydroxy-dihydropleurotinic acid, obtained as a bright yellow solid with a molecular formula of C 21 H 24 O 6 as well. The 1 H and 13 C-NMR data resemble largely those of compound 5, with the major difference being two methines at δ C/H 63.9/4.55 and 32.4/2. The molecular formula C 21 H 26 O 5 of leucopleurotinic acid (7), a white amorphous powder, was retrieved from HRESIMS, entailing nine degrees of saturation. Again, NMR data largely resembled those of dihydropleurotinic acid (3) with a major difference being upfield shifts in the 13 C-NMR data of the olefinic carbons, suggesting a reduction of the quinone moiety to its corresponding hydroquinone form.
Closely related to 7 is metabolite 8, 14-oxo-leucopleurotininc acid, acquired as an off-white amorphous powder and a molecular formula of C 21 H 24 O 6 . NMR spectral data coincide to great extent with compound 7, yet indicate a keto group at position C-14 (δ C 202.7).
A light brown amorphous powder with a molecular formula of C 21 H 22 O 5 and eleven degrees of saturation is nematoctone (9). Its 1 H and 13 C-NMR data ( Table 2) overlap in many parts with 8, yet an 1 H, 13 C-HMBC correlation of C-13 to H-14 (δ H 5.64) portend an ester bond between C-13 and C-14 (δ C 74.9), generating leucopleurotin as the underlying core structure. However, the usual chemical shifts of C-4 and C-12 were not observed and instead carbon signals at δ C 110 (C-12) and 147.7 (C-4) showed up. 1 H, 13 Figure S2).
Di-oxo-leucopleurotinic acid (10) was isolated as a light brown powder. Its molecular formula was determined by HRMS as C 21 H 24 O 7 and ten degrees of saturation. Compared to 8, the molecular formula includes an additional oxygen atom. A closer look at the 13 C spectra showed the appearance of a second keto group at δ C 205.3 (C-15) in proximity to H-1 and H-9 ( 1 H, 13 C-HMBC; Supplementary Materials) and an upfield shift of C-11 to δ C 67.4. This suggested that the seven-membered cyclic ether was cleaved, as compared to 9.  A carbon at δ C 74.8 (C-8) was observed instead of a methine, indicating a hydroxy function attached to C-8, and HMBC correlations of C-7 and C-9 to a methylene at δ C 63.9 (C-13) suggested the proximity of another hydroxyl function. Another carbon at δ C 72.8 (C-4), carrying an OH, showed correlations in the 1 H, 13 C-HMBC to two methyl groups at δ H 1.18 (H-11) and 1.26 (H-12), leading to a 4-ring system as the underlying core structure, similar to 10. This scaffold was confirmed by 1,1-ADEQUATE NMR data (measured in DMSO-d 6 , see Supplementary Materials). The stereochemistry of C-8 was assigned to be R by ROESY correlations of 8-OH to H-9, as well as a strong correlation of H-13 to H-15 (see Supplementary Materials), indicating an axial orientation of C/H 2 -13. The axial orientation of C/H 2 -13 was further confirmed by a J-HMBC NMR experiment, since a large coupling constant of 6.1 Hz was observed between H-7 ax (δ H 1.23) and C-13 indicating an anti-periplanar orientation between H-7 ax and C-13 and thus confirming the 8R configuration, giving the absolute configuration of 4-hydroxypleurogrisein 3S,5S,8R,9R,10R.

Results and Discussion
As reported earlier, the producing strain MFLUCC 12-0451 was identified as Hohenbuehelia grisea (Peck) Singer [12]. H. grisea, more commonly known under its former name of the asexual morph Nematoctonus robustus, has been reported to produce pleurotin (1), dihydropleurotinic acid (2), and leucopleurotin (3) [5]. The group of Arigoni (ETH Zurich) has in the past extensively studied the biosynthesis of pleurotin and in the course of their research proposed several structures [13], which are also being reported with full NMR spectral data in this study for the first time.
3-Hydroxy-dihydropleurotinic acid (5) was isolated as an off-white to pale yellow powder with a molecular formula of C21H24O6, retrieved from a molecular ion cluster [M + H] + at m/z 373.1661, indicating ten degrees of saturation. The correlation of 1 H and 13 C-NMR data (Table 1) in combination with HMBC correlations (Figure 3) led to the establishment of dihydropleurotinic acid as the underlying core structure [5,12], with the novelty of compound 5 being a hydroxyl functionality at C-3 (δC 82.8), which leads to the absolute configuration of 3S,4S,5S,8R,9S,10R,15S.
Next, the compounds were tested for their inhibitory effect against hepatitis C virus (HCV). HCV infections is continuing to impose a global threat to human health with 71 million people infected worldwide. Although various potent direct acting antiviral agents have been licensed, high costs prevent the majority of infected individuals from having access to treatment. Out of the compounds tested, only 4-hydroxypleurogrisein (11) showed significant activities in vitro, while compound 5, which was tested concurrently, was devoid of any activity on the host cells. As depicted in Figure 4, HCV infectivity decreased in a dose-dependent manner with an IC 50 value of around 5 ng/µL and strong inhibitory effect at 10 ng/µL, while at 20 ng/µL cytotoxic effects were noted. The green tea molecule epigallocatechin gallate (EGCG) was used as positive control [14]. Table 3. Minimum inhibitory concentration (MIC) in the serial dilution assay for bacteria and fungi and half-inhibitory concentrations (IC 50 for cell lines) in µg/mL. For determination of MICs, 20 µL of either 1 mg/mL stock solution (67 µg/mL) or 1.5 mg/mL (100 µg/mL) of 1-3, 5-11 were tested. Cell density was adjusted to 6.7 × 10 5 cells/mL. Twenty microliters of Ethanol were used as negative control and displayed no activity against the selected test organisms. For IC 50 values, 6 × 10 3 cells/well were sown in 96-well microtiter plates and treated with 1-3, 5-11 over five days. compound 5, which was tested concurrently, was devoid of any activity on the host cells. As depicted in Figure 4, HCV infectivity decreased in a dose-dependent manner with an IC50 value of around 5 ng/µL and strong inhibitory effect at 10 ng/µL, while at 20 ng/µL cytotoxic effects were noted. The green tea molecule epigallocatechin gallate (EGCG) was used as positive control [14].  The inoculum was removed 4 h later and monolayers were washed three times with PBS and overlaid with fresh medium containing no inhibitors. Infected cells were lysed three days later, and reporter virus infection was determined by renilla luciferase activity (top). The cell viability was measured by determination of firefly luciferase (bottom), which is stably expressed in the target cells.

Fungal Material
Basidiomes of the nematode-trapping fungus of H. grisea were collected from decaying wood in the tropical rainforest of Thailand near the Mushroom Research Centre, Chiang Mai Province, Thailand (http://www.mushroomresearchcentre.com/), in August of 2012 and the corresponding culture was obtained from basidiospores. The dried specimen and a corresponding culture are deposited at the mycological herbarium of the Mae Fah Luang University Culture Collection, Chiang Rai, Thailand, under the accession number MFLUCC 12-0451. Its 5.8S gene region, the internal transcribed spacer 1 and 2 (ITS) and part of the large subunit (LSU) were previously sequenced and published by Sandargo et al. [12] and the sequence data are deposited with GenBank, accession number MF150036.

Fermentation and Extraction
The strain H. grisea MFLUCC 12-0451 was cultivated in two different liquid media BAF (DSMZ 392), and Robbins medium [4] modified based on results of Shipley et al. [9] using alder extract (100 g Alnus glutinosa dead wood and branches without leaves, collection site Helmholtz Centre for Infection Research, campus Braunschweig, Germany, soaked for 24 h at room temperature in 1 L of deionized water)]. A sufficiently grown culture of H. grisea MFLUCC 12-0451 on BAF (DSMZ 392) agar was utilized to inoculate 200 mL of each medium in 500 mL Erlenmeyer flasks, incubated on a rotary shaker at 24 • C and 140 rpm. After 14 days, 30 mL of these seed cultures were shifted into 6 × 2 L Erlenmeyer flasks with 800 mL of the respective medium each. Incubation of the cultures at 24 • C and 140 rpm on rotary shakers occurred until all free glucose was consumed, after 21 days in BAF [12] and 24 days in modified Robbin's media. Extraction followed an earlier published protocol, described in Sandargo et al. (2018) [12], leading to 4 g of crude extract for BAF medium and 2 g for modified Robbins medium. All crude extracts were filtered using an SPME Strata™-X 33 u Polymeric RP cartridge (Phenomenex, Inc., Aschaffenburg, Germany).

Antimicrobial Activities
The minimum inhibitory concentration (MIC) for each compound was ascertained in a serial dilution assay in 96-well microtiter plates, as previously published by Kuhnert et al. [15], using YM media for yeasts and filamentous fungi and BD TM Difco™ Mueller Hinton Broth for bacteria.

Cytotoxicity Assay
In vitro cytotoxicity assay was performed as described by Richter et al. [16] against the mouse fibroblast cell line L929 and the cervix carcinoma cell line KB3.1.

Inhibitory Effects on HCV Infectivity
This assay was carried out as described previously by Mulwa et al. [18]. Huh7.5 cells stably expressing Firefly luciferase (Huh7.5 Fluc) were cultured in Dulbecco's modified minimum essential medium (DMEM, Life Technologies, Darmstadt, Germany) containing 2 mM L glutamine, 1 × minimum essential medium nonessential amino acids (MEM NEAA, Life Technologies), 100 µg/mL streptomycin, 100 IU/mL penicillin (Life Technologies), 5 µg/mL blasticidin and 10% fetal bovine serum. Cells were maintained in a 37 • C environment with 5% CO 2 supply. Cells were infected with Jc1-derived Renilla reporter viruses in the presence or absence of compounds as described previously [19]. Infected cells were lysed and then frozen at −80 • C for 1 h following measurements of Renilla and Firefly luciferase activities on a Berthold Technologies Centro XS3 Microplate Luminometer (Bad Wildbad, Germany) as indicators of viral genome replication and cell viability, respectively.
Supplementary Materials: The following are available online. Table S1: Retention time of isolated peaks and gradients used for preparative HPLC, water agar test with C. elegans, Figure S1: Nematode captured by H. grisea, HRESIMS data of 5-11, and full NMR data of 5-11.