Cytotoxic and N-Acetyltransferase Inhibitory Meroterpenoids from Ganoderma cochlear

Seven compounds, including two pairs of new meroterpenoids, (+)- and (−)-gancochlearol C (1), (+)- and (−)-cochlearoid Q (3), and a new meroterpenoid gancochlearol D (2), together with four known meroterpenoids were isolated from the aqueous EtOH extract of the fruiting bodies of Ganoderma cochlear. Their structures were determined by spectroscopic data. The isolated compounds were evaluated for their cytotoxic activity against three human lung cancer cells (H1975, PC9, A549) and N-acetyltransferase inhibitory property. The results show that (+)-gancochlearol C could inhibit N-acetyltransferase with an IC50 value of 5.29 μM. In addition, ganomycin F was found to show moderate activity against the H1975 human lung cancer cell line, with an IC50 value of 19.47 μM.


Introduction
Ganoderma fungi (Ganodermataceae) have long been used as medicines in China, Japan and other Asian countries [1]. Pharmacological investigations revealed that Ganoderma extracts or compounds possess antitumor [2], immunomodulatory [3], antioxidant [4], and anti-inflammatory properties [5]. Mounting chemical investigations have been underwent on Ganoderma in the past decades which mainly focused on the isolation of triterpeniods and polysaccharides. In 2013, we identified a pair of novel meroterpenoids named lingzhiols with a 5/5/6/6 ring system from Ganoderma lucidum as a p-Smad3 inhibitor [6], which received great attention in the related scientific community. So far, more than 150 Ganoderma meroterpenoids have been reported from Ganoderma species, of which more than two thirds were characterized by us. These structurally diverse meroterpenoids were disclosed to have anti-fibrotic [7,8], neuroprotective [9], antiinflammatory, and antioxidant properties [10][11][12][13][14][15][16]. As a continuation of our research on Ganoderma species, Ganoderma cochlear was investigated which resulted in the isolation of cochlearines A and B, cochlearoids A-K [8,16], cochlearols A and B [17], and ganocochlearines C-I [18]. These findings inspired us to conduct an additional investigation on this fungus, which therefore led to the isolation of new meroterpenoids, gancochlearol C (1), cochlearoid Q (3), and gancochlearol D (2), together with four known ones, ganomycin C (4), ganomycin F (5), cochlearol D (6), and fornicin D (7). In this paper, we describe their isolation, structural elucidation as well as biological activities toward human lung cancer cells (H1975, PC9, A549) and N-acetyltransferase.

Biological Evaluation
All the isolated meroterpenoids were evaluated for their cytotoxic activity against three human lung cancer cell lines (H1975, PC9, A549). The results show that compounds 2 and 5 are cytotoxic against A549 cells at 40 µM, with 5 shown to be the most active ( Figure S46). Besides, 2 and 5 are also found to be active against H1975 and PC9 cells at 40 µM. With this, the cytotoxic potency of 2 and 5 toward three cancer cell lines were further determined using the CCK-8 assay. As shown in Table 4, compound 2 inhibits three cancer cells (H1975, PC9, A549) with IC 50 values of 32.43, 40.57, and 30.65 µM, respectively. However, compound 5 appears to be more potent than 2 with IC 50 values of 19.47, 35.70, and 21.60 µM, respectively, against H1975, PC9, and A549 cells. In this assay, erlotinib was used as the positive control with the IC 50 values of 7.66, 0.085, and 4.59 µM, respectively, toward the above mentioned cells. Arylamine N-acetyltransferase (NAT) was reported to be related with synthesis of mycolic acids and complex lipids in Mycobacterium bovis, which makes NAT a novel drug target for tuberculosis [24]. With this, all the isolates except for compounds (+)-3 and (−)-3 were tested for their inhibitory activity toward the recombinant NAT2 isoform. It was found that (+)-1 and (−)-1 could inhibit NAT2 at 10 µM, and (+)-1 is more potent than quercetin, which was the positive control used in this study. Based on this observation, a subsequent experiment was carried out to reveal that (+)-1 has an IC 50 value of 5.29 ± 0.10 µM toward NAT2 (Figure 4).

Fungal Material
The fruiting bodies of Ganoderma cochlear were purchased from Tongkang Pharmaceutical Co. Ltd. in Guangzhou, Guangdong Province, China, in July 2014. The material was authenticated by Prof. Zhu-Liang Yang at Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, China, and a voucher specimen (CHYX-0589) has been deposited at School of Pharmaceutical Sciences, Shenzhen University Health Science Center, Shenzhen, China, since October 2017.

Extraction and Isolation
The powders of Ganoderma cochlear (200 kg) fruiting bodies were extracted with 80% EtOH under reflux (3 × 120 L, 4, 3, 3 h) and concentrated under reduced pressure to yield a crude extract. An aliquot (8 kg extract corresponding to 95 kg fungal material) was suspended in water and partitioned with EtOAc thrice, followed by removal of solvents to afford an EtOAc soluble extract (4 kg). The extract was separated by silica gel column with increasing acetone in petroleum ether to provide four parts (Fr.1-Fr.4) Table 1.  Table 2.  Table 3.

Cell Viability
The human NSCLC cell line (NCL-H1975) was purchased from Shanghai Cell Bank of Chinese Academy of Sciences, (Shanghai, China). PC9 and A549 were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA). Cells were cultured in DMEM (Gibco, Grand Island, NY, USA) with 4.5 g/L glucose and 10% FBS (Gibco, Grand Island, NY, USA). Erlotinib used in the experiments were purchased from Selleck (Selleck Chemicals, Houston, TX, USA). Cytotoxicity was determined using the previously described method [25]. Three human lung cancer cell lines were incubated in 96-well plates (5000 cells/well) respectively to attach overnight and then exposed to drug treatments for additional 24 h. And the CCK solution was added, after 2 h of incubation, the absorbance was measured at a wavelength of 450 nm. Cell viability was measured by a TransDetect ® Cell Counting Kit (TransGen Biotech, Beijing, China). The IC 50 values were calculated using the GraphPad Prism 5.0 (GraphPad Software, San Diego, CA, USA).

N-Acetyltransferase (NAT) Inhibition
In order to determine the inhibitory activity of the isolates toward N-acetyltransferase (NAT), which plays an important role in the synthesis of mycobacterial cell wall lipids and is thus closely associated with mycobacterial growth, the recombinant NAT2 (Corning Company, Corning, NY, USA, Cat. No. 456282), a key target for the inhibition of Mycobacterium tuberculosis was utilized in this study. The inhibition activity of the compounds toward NAT2 were assayed according to the previously reported method [26]. Briefly, the fluorescent probe (CYP1) was incubated with NAT2 in the presence or absence of different compounds including (+)-1, (−)-1, 2, and 4-7 with the final concentration of 10 µM. After incubation for 30 min, the reaction was terminated by adding acetonitrile and the supernatant was tested by Synergy H1 microplate reader (Bio-Tek, Winooski, VT, USA) with the excitation wavelength at 780 nm. Meanwhile, the positive inhibitor quercetin (10 µM) for NAT2 was also set in this inhibition screening [27]. Finally, the IC 50 value of compound (+)-1 was further investigated by adding different concentrations of (+)-1 (0.5−10 µM) in the above incubation system. Data were fit to log (inhibitor) versus normalized response-variable slope equation using GraphPad Prism 6 (GraphPad Software, San Diego, CA, USA).

Conclusions
In sum, three new meroterpenoids were characterized from the fruiting bodies of Ganoderma cochlear which adds new facets to the structural diversity to the family of Ganoderma meroterpenoids. Our findings of cytotoxic meroterpenoids suggest that this class of compounds might justify medicinal applications of Ganoderma fungi in cancer. Finally, inhibition of (+)-1 toward NAT2 implies that it could be a structure template for developing NAT2 inhibitors with therapeutic potential in tuberculosis.