1. Introduction
Lichens are complex life forms in which algae and fungi maintain a symbiotic relationship. They not only live in extreme environments, such as tundra, desert, and volcanic areas in the polar regions, but also grow in various places, such as rainforests and temperate regions [
1]. The primary and secondary metabolites of lichens have excellent pharmacological effects, such as anti-cancer, anti-inflammatory, osteoporosis relief, and anti-biotic, and thus research to identify source materials for new drug development has been underway [
2,
3]. The species of
Thamnolia vermicularis (Sw.) Schaer. exist in a variety of environments, such as gravel, frost boils tundra, and mossy bushes [
4]. The polysaccharide extract of this lichen has shown various biological activities such as antitumor and immunomodulatory activities [
5].
On the other hand, Alzheimer’s disease (AD) is the most common degenerative brain disease that causes dementia [
6]. It develops very slowly and is characterized by a gradual progression. In the early stages, it mainly causes problems with memory for recent events and is accompanied by other cognitive abnormalities, such as speech and judgment, eventually losing all daily life functions [
7]. In AD, an excessive accumulation of β-amyloid is known as a key mechanism [
8], and the activities of monoamine oxidase (MAO) and cholinesterases (ChEs) induce AD [
9]. The number of patients with AD is increasing each year worldwide; therefore, it is important to identify an effective way to treat AD. In addition, depression is a phenomenon in which depressed mood, interest, and enjoyment decrease, resulting in discouragement, hopelessness, and sometimes suicidal thoughts [
10]. It is known that the reduction of neurotransmitter monoamines in neuronal synapses, such as norepinephrine and serotonin, is the main cause of depression, MAO breaks down monoamines and causes depression [
11].
MAO is an enzyme that catalyzes the oxidative deamination of neurotransmitter monoamines and has two isoforms, A and B [
12]. MAO-A is associated with neuropsychiatric disorders, such as depression, and MAO-B is related to neurodegenerative diseases, such as AD and Parkinson’s disease (PD) [
13]. ChEs are classified into acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). AChE breaks down acetylcholine (ACh) into acetate and choline [
14]. ACh is most widely distributed in the cerebral cortex and is known to be a representative neurotransmitter. AD patients appear to be deficient in the amount of ACh in the brain. BChE breaks down butyrylcholine, and the level of BChE is high in the cerebrum of AD patients [
15,
16]. In addition, β-site amyloid precursor protein cleaving enzyme 1 (β-secretase, BACE-1) affects the progression of AD [
17,
18].
In this study, the inhibitory activities against MAO-A, MAO-B, AChE, and BChE for a library of 195 extracts of endogenous lichen fungi (ELF) were evaluated as well as antioxidant activity, and 5-hydroxy-2-methyl-chroman-4-one (HMC) was isolated from an extract of ELF13, identified as Daldinia fissa, as a strong MAO-B inhibitor. In addition, an inhibitory activity for BACE-1, kinetic studies, cytotoxicity tests, in silico pharmacokinetics, and docking simulations were carried out for the compound HMC.
2. Materials and Methods
2.1. Extracts of Endogenous Lichen Fungi and Evaluations of Inhibitory Activities
A library of 195 extracts with ethyl acetate (EA) or butanol (BuOH) of endogenous lichen fungi (ELF) derived from Ukraine was obtained from Korea Lichen Research Institute (KoLRI) in Sunchon National University, Republic of Korea. The extracts were tested for their inhibitory activities against MAO-A, MAO-B, AChE, and BChE, and their antioxidant activity. All chemicals and enzymes were purchased from Sigma-Aldrich (St Louis, MO, USA), unless otherwise specified.
2.2. Monoamine Oxidase (MAO) Activity Assay
Human recombinant MAO-A and MAO-B enzymes were used, and the activities were measured in a final volume of 500 µL of 100 mM sodium phosphate buffer of pH 7.2, and 0.06 mM kynuramine (MAO-A), or 0.3 mM benzylamine (MAO-B). MAO-A and MAO-B activities were assayed at 316 nm and 250 nm, respectively, for 30 min in the kinetic mode [
19,
20].
2.3. Cholinesterase Activity Assay
For cholinesterase activity assay, electric eel AChE and horse serum BChE were used. Activities were measured according to the Ellman method [
21], with slight modifications [
22,
23], in 50 mM sodium phosphate buffer (pH 7.5). After adding cholinesterase enzymes and the inhibitor, pre-incubation was applied for 15 min. After adding 0.5 mM 5,5-dithiobis (2-nitrobenzoic acid) (DTNB) 0.5 mM for color development, 0.5 mM acetyl thiocholine iodide (ATCI) or 0.5 mM butyrylthiocholine iodide (BTCI) was used as a substrate. The activities were measured in a final volume of 500 µL in the kinetic mode for 15 min at a wavelength of 412 nm.
2.4. β-Site Amyloid Precursor Protein Cleaving Enzyme 1 (BACE-1) Activity Assay
BACE-1 assay was carried out using the BACE-1 activity detection kit and a fluorescence spectrometer (FS-2, Scinco, Seoul, Korea) by an excitation at 320 nm and an emission at 405 nm, after the reaction for 2 h at 37 °C [
24].
2.5. Antioxidant Activity Assay
The antioxidant activity was measured at 100 µg/mL of the extracts using 0.1 mM 2,2-diphenyl-1-picrylhydrazyl (DPPH). After 15 min of pre-incubation, absorbance was measured at 517 nm [
25].
2.6. Culture and Extraction of the Selected Endogenous Lichen Fungi
The selected ELF were cultured in potato dextrose broth (PDB) agar medium. The cultured fungi were inoculated into 200 mL of PDB liquid medium by adding 5 × 5 mm (3–4 pieces) and incubated at 20 °C and 150 rpm for 2 weeks. The culture solution was suspended with a homogenizer (HG-15A, Daihan Sci. Co., Wonju, Korea) and extracted with the same volume of EA for 2 h at 20 °C and 150 rpm. The supernatant was filtered through a Whatman No. 1 filter, then concentrated, and recovered through a vacuum rotary concentrator.
2.7. Thin-Layer Chromatography
Thin-layer chromatography (TLC) was performed to separate constituents in the extract using Prep TLC plates (PTLC Silica gel 60 F254, 0.5 mm, Merck, Darmstadt, Germany). The extract was developed, and the spots on the chromatography were recovered under 254 nm through an activity-guided method. The first solvent was ethyl acetate and toluene in a ratio of 1:9 (v/v), and the second solvent was chloroform and toluene in a ratio of 1:9 (v/v). The concentration of the sample used was 100 mg/mL, and a maximum volume of 500 µL was loaded onto the PTLC plate.
2.8. Structure Analysis through Nuclear Magnetic Resonance (NMR), Liquid Chromatography–Mass Spectrometry (LC-MS)
Nuclear magnetic resonance (NMR) spectra were recorded with an NMR spectrometer (Varian Medical Systems, Inc., VA, USA) at 400 MHz for 1H and 100 MHz for 13C in MeOD using a solvent signal as an internal reference (δH 4.870/δC 49.150). Optical rotations were acquired using a polarimeter (Optronic P-8000, Kruss, Hamburg, Germany) with a 5 cm cell.
2.9. Inhibitory Activities of the Compound and Enzyme Kinetics
The inhibitory activities of the isolated compounds were analyzed at a concentration of 2 µg/mL. After measuring the IC
50 value of the compound, the K
i value was determined using Lineweaver-Burk plots at ~1/2 × IC
50, IC
50, and 2 × IC
50 concentrations, and a secondary plot obtained by the slope versus the inhibitor concentrations [
23].
2.10. Inhibitor Reversibility Analysis
The reversibility analysis of the compound was performed by a recovery experiment through dialysis using 100 mM sodium phosphate (pH 7.2) buffer at 2 × IC
50 [
26]. For the reversibility analysis of MAO-B, the reversible inhibitor lazabemide and the irreversible inhibitor pargyline were used as references.
2.11. Cytotoxicity Analysis of the Compound
Cell viability was determined according to the cell counting kit (CCK)-8 assay method [
27], using MDCK (Madin–Darby canine kidney) and HL-60 (human acute promyelocytic leukemia) cells, as normal and cancer cells, respectively. Briefly, MDCK and HL-60 cells were resuspended in Dulbecco’s modified eagle medium (DMEM) and Roswell Park Memorial Institute (RPMI)-1640, respectively, at 1 × 10
5 and 3 × 10
5 cells/mL, respectively. The cell suspension (100 µL) was added to each well of the 96-well plate and was incubated for 24 h at 5% CO
2 and 37 °C. After incubation, 100 µL of the medium supplemented with 1, 3, 10, 30, and 50 µM of the compound was added to each well and incubated again. After 24 h, 100 µL of the solution was removed from each well, and CCK-8 (10 µL/well) was dispensed. After 2~4 h, the absorbance was detected at 450 nm with a microplate reader (Versamax, Molecular Devices, CA, USA).
2.12. Docking Simulations and Molecular Dynamics of the Compounds with Monoamine Oxidase-A (MAO-A) and MAO-B
To simulate the dockings of (
S)- or (
R)-HMC to MAO-A or MAO-B, AutoDock Vina plugin in UCSF Chimera 1.14 (build 42094) was used, which has an automated docking facility [
28,
29]. To define the docking sites of hMAO-A and hMAO-B, the predefined active sites in MAO-A/7-methoxy-1-methyl-9H-beta-carboline (HRM) complex (PDB ID: 2Z5X) [
30] and MAO-B/pioglitazone complex (P1B) (PDB ID: 4A79) [
31] were used. To prepare target proteins, all molecules including water except the flavin-adenine dinucleotide (FAD) were removed from the target structures, and then hydrogens and charges were added. For the docking simulation, the 2D structures of the compounds were created and converted into 3D structures, and energy minimization was conducted with the ChemOffice 2002 (ChembridgeSoft). Based on the results of the docking simulation, we checked for possible hydrogen bonding using the bonding relaxation constraints of 0.4 Å and 20.0° using Chimera [
32]. The amino acids within 4Å from the docked poses were depicted as key residues, and the FAD was also displayed together to show the distances from the docked compounds. We performed molecular dynamics of HMC enantiomers complexed with MAO-A and MAO-B using NAMD 2.21 [
33] and VMD 1.9.4 [
34] software, and applied the CHARMM 36 parameters to the analysis at CHARMM-GUI website (
http://www.charmm-gui.org/) [
35], with 10,000 steps as the initial minimum at 310K. The data were examined using the root mean square deviation (RMSD) by time and the structure variation was calculated by RMSD values of protein-ligand complexes from 0 to 1000 ps.
2.13. Pharmacokinetic Analysis of the Compound Using the In Silico Method
The pharmacokinetic and physicochemical analysis of the compound was performed using the web tool of SwissADME (
http://www.swissadme.ch/), and gastrointestinal absorption, blood-brain barrier permeability, P-glycoprotein substrate, and cytochrome P450 inhibitory activities were analyzed [
36].
4. Discussion
In this study, the inhibitory activities of 195 ELF extracts from Ukraine-derived lichens against MAO-A, MAO-B, AChE, and BChE were evaluated as well as their antioxidant activities. Among them, several effective extracts were selected: two extracts for MAO-A, five for MAO-B, two for AChE, one for BChE inhibitory activities, and three for antioxidant activity. Among them, ELF13 extract showed the highest inhibitory activity against MAO-B. ELF13 was an endogenous fungus Daldinia fissa forming a symbiotic relationship with the lichen Thamnolia vermicularis (Sw.) Schaer.
Extracts and compounds of lichens were shown to have effective inhibitory activities against various enzymes, including MAO and AChE in a recent review [
39]. However, little information is available: Solorinic acid of anthraquinones inhibited MAO enzyme with IC
50 14.3 µM [
40]. A synthetic derivative 4-acylresorcinol displayed potent inhibitory activity with IC
50 value 4.27 µM, among lichen compounds and their synthetic analogues [
41]. The inhibitory activities were determined using liver or brain MAO and the IC
50 values of the compounds were higher than that of HMC, which showed strong and ~4-fold selective MAO-B inhibitory activity (IC
50 = 3.23 µM).
HMC is known to have several biological activities, such as antifungal and phytotoxicity [
42], and an inhibition of
Saccharomyces cerevisiae growth and the formation of soybean callus [
37]. Mellein, an isochromane, is known to inhibit monoamine oxidase [
43], HCV protease [
44], hepatitis C virus protease [
45], and human DNA polymerase lambda [
46]. The potency of HMC for MAO-B is much higher than that of mellein (IC
50 = 8.93 µg/mL, 50.16 µM)) [
43].
Lazabemide (IC50 = 0.063 µM) is a reversible inhibitor of MAO-B and is used in the treatment of PD and AD. The potency of HMC is lower that of lazabemide; however, it can be served as a lead compound or a scaffold for the development of promising derivatives through the large-scale cultivation of fungi as a natural compound.
The docking simulations revealed that HMC interacted with the Cys172 of MAO-B to form a hydrogen bond, while no hydrogen bond interaction with MAO-A was predicted. Therefore, hydrogen bond interaction might play an important role in the strong binding and inhibitory activity of HMC against MAO-B. Although the positions and orientations of two (S)- and (R)-enantiomers at the MAO-B active site were different, few differences in binding affinities were predicted. Collectively, binding affinity of HMC to MAO-B is greater than that of MAO-A, and the affinities of (S)-enantiomer to the enzymes are comparable to those of (R)-enantiomer. Molecular dynamics also supported the experimental data and the docking simulations well. In addition, HMC crosses the blood–brain barrier and shows no violations of Lipinski’s rule of five, and no intracellular toxicity, indicating pharmacological potential. This indicates that HMC can be considered a candidate for the treatment of neurodegenerative diseases.
5. Conclusions
HMC, isolated from an ELF extract, showed a strong inhibitory activity against MAO-B (IC50 = 3.23 µM) with a moderate selectivity over MAO-A (IC50 = 13.97 µM), and is a reversible competitive inhibitor of MAO-B. HMC bound to MAO-B with a binding energy of −7.3 kcal/mol, which was greater than its affinity with MAO-A (−6.1 kcal/mol), indicating that HMC is a more potent and selective inhibitor for MAO-B than MAO-A. In addition, HMC showed pharmacological advantages as it has high gastrointestinal absorption, passes through the blood–brain barrier, and is non-toxic. The results in this study suggest that ELF can be an excellent resource in exploring new pharmaceuticals.