Characterizing novel anti-oncogenic triterpenoids from ganoderma

The fungus Ganoderma lucidum is a traditional Chinese medicine that has been used for centuries as a nutritional supplement and herbal medication.¹ Over 4 decades of research have revealed over 430 secondary metabolites isolated from diffrerent species of Ganoderma. Over 150 lanostane-type triterpenoids have been reported in Ganoderma lucidum. These lanostane triterpenoids purified from Ganoderma lucidum are thought to be the main active compound underlying preclinical reports of anti-cancer function.^2-4 There studies provide detail protocol for the systematically extracting triterpenoids from Ganoderma lucidum and for evaluating their antitumor activities.

In our recent study, we purified 30 known and 5 novel lanostane-type triterpenoids from the fruiting bodies of Ganoderma luncidum.³ Briefly, the dry fruiting bodies of Ganoderma lucidum were extracted by ethyl acetate. The ethyl acetate crude extract were then incubated in chloroform to obtain total triterpenoids. This step removed water soluble molecules including the polysaccharides, which are another fraction possessing anti-tumor properties.⁵ With CHCl₃ fractionation and repeated column chromatography in preparative High Performance Liquid Chromatography (HPLC), 35 compounds were purified. These compounds were identified by physicochemical properties and spectral data analysis (Table 1). Previous studies have indicated that Ganoderma triterpenoids could modulate cell cycle progression, cell proliferation, cardiovascular functions and tumor growth.^2,6,7 Our study demonstrated that the novel compounds, annotated 1, 2 and 3 could inhibit cancer cell proliferation and survival, and compounds 4, 7, 20 and 24 also possessed antitumor activities.³

Table 1.

Name of 35 compounds isolated from Ganoderma lucidum.

Number	Compounds name
1*	ganodermanontriol
2*	3β, 24S, 25R, 26- tetradroxy-7α- methoxy-8- ene-lanost-ol
3*	12α- methoxy-ganodermanondiol
4*	15β- hydroxy-lucidumol A
5*	15α- hydroxy-ganodermanontriol
6	ganoderiol D
7	ganoderiol F
8	ganoderiol B
9	ganoderiol E
10	ganoderic acid β
11	ganoderic acid A
12	ganoderic acid B
13	ganoderic acid C
14	ganoderic acid D2
15	12β-hydroxy-3,7,11,15,23-pentaoxo-lanosta-8-en-26-oic acid
16	ganodermanontriol
17	11- trione-24(S), 25- dihydroxy-lanosta- 8- ene
18	ganoderitriol M
19	lucidumol A
20	lucidadiol
21	ganoderiol A
22	24R, 25S, 26- trihydroxy-lanosta- 7, 9(11) - dien- 3- one
23	lucidumol B
24	ganodermanondiol
25	lucidenic acid A
26	ganolucidic acid A
27	ganoderic acid J
28	methyl lucidenate A
29	ganoderic acid E
30	ganoderenic acid d
31	ganoderic acid C2
32	ganoderic acid F
33	ganoderic acid G
34	ganoderic acid H
35	ganoderic acid AM

*New compounds.

We used 3D-QSAR software for in silico correlation of structure and activity. This software was used to provide theoretical guidelines to identify active groups within the chemical compounds. This software could also be used to modify chemical structure to design novel molecules. In our study, 17 compounds (1–9, 17, 19–24 and 28) were studied in detail using 3D-QSAR. Quantitative structure-activity relationships were built using the 3D-QSAR protocol in Discovery Studio 3.5 (Accelrys, Co. Ltd.). According to the training set (ca. 75% of the data set) and the test set (ca. 25% of the data set), a subset of 13 compounds was used as a training set for QSAR modeling. The results (correlation coefficient r² = 0.968) demonstrated the stability and reasonable predictability of our 3D-QSAR protocol. In this experiment, a comparative analysis of the actual vs predicted IC50 values confirmed that this model was reliable in forecasting biochemical activity for Ganoderma triterpenoids. Comparing the structure and activities of compounds 9, 21 and 23 (IC50 ≈ 160 μM) with compound 3 (IC50 = 21.2 μM), we found that the introduction of a methyl group of steric hindrance at C-2 and negative O atom such as carbonyl group at C-7 to replace neutral hydrogen atoms may decline biochemical activity of compounds 9, 21 and 23.³ The chemical structures of these molecules are provided in the original publication.³

To understand better the mechanism of compounds 3 which displayed the highest activity against cancer cell growth, we used the Pharmaceutical Target Seeker (PTS, http://www.rcdd.org.cn/PTS/index) to further predict its target site. The data showed that compound 3 could target tumor necrosis factor (TNF-α). The interactions of compound 3 with TNF-α was further analyzed with molecular docking using the Discovery Studio as implemented through the graphical user interface CDOCKER protocol. The docking results revealed that the amino acid Tyr119 located in the binding pocket of the protein connected to compound 3 by one hydrogen bond.

The 5 new triterpenoids we extracted expanded the chemical library and may possibly provide promising leads of developing anti-cancer agents. The 3D-QSAR and cytotoxicity results revealed the relationship of structure and function of the compounds. For example, compound 3 that possesses the highest activity has a potential anticancer effect by targeting TNF-α, an important molecule in cancer progression. Our work thus provides some guidelines to design and optimize the synthesis of anticancer compounds based on the triterpenoids from Ganoderma lucidum. This will be followed by examination of the molecular mechanisms underlying this effect. Based on these results, our next step is to design and synthesize promising novel compounds with high anticancer activity.

Funding Statement

Our studies were supported by the High-level Leading Talent Introduction Program of GDAS (No.2016GDASRC-0102, 2017GDASCX-0820), Guangdong Province Natural Science Foundation (No.2015A030313711, 2017A030310088), Guangdong Province Science and Technology Project (No.2016A030303041).

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.