Identification of novel phytocannabinoids from Ganoderma by label-free dynamic mass redistribution assay
Graphical abstract
Introduction
Cannabinoid receptors (CB1 and CB2) belong to the G-protein coupled receptor (GPCR) superfamily and they are primarily coupled to inhibitory G proteins (Gi) (Howlett, 2002). They are part of the endocannabinoid system (ECS) named after the plant Cannabis sativa L. (Gaoni and Mechoulam, 1964). The ECS is a complex lipid signaling and immunomodulator system, and it is probably the most important physiologic system. Alterations of the ECS are reported to be associated with neuropsychiatric disorders and immune vulnerability (Pacher et al., 2006; Zoppi et al., 2011; Zou and Kumar, 2018). CB1 is expressed at high levels in the brain (Matsuda et al., 1990), playing a role in memory (Hebert-Chatelain et al., 2016), sleep (Murillo-Rodriguez, 2008), mood (Witkin et al., 2005), and pain management, and its agonists show neuroprotective properties (Molina-Holgado et al., 2005). CB2 receptor localization indicates a high density in immune cells (Munro et al., 1993), and multiple pathologic conditions can be mediated by CB2 activation (Atwood et al., 2012a), such as multiple sclerosis (Chiurchiu et al., 2018), HIV infection (Purohit et al., 2014), and Alzheimer's disease (Ramirez et al., 2005). CB2 stimulation is important for development of neuroprotective therapies (Lourbopoulos et al., 2011; Merighi et al., 2012). The involvement of cannabinoid receptors in health and diseases is currently a topic of interest (Pacher and Kunos, 2013).
Apart from endocannabinoids, exogenous ligands can induce effects through the receptors as well. Nature serves as a reservoir full of active ingredients with rich structural diversity. So far, more than 100 cannabinoids (also known as phytocannabinoids) have been identified from the Cannabis sativa L., among which Δ9-tetrahydrocannabinol (Δ9-THC) is the principal psychoactive component (Fig. 1) (Gaoni and Mechoulam, 1964). Naturally occurring components are an important source of novel drugs and therapies. For example, HU-210 is developed based on the structure of Δ8-THC with enhanced affinity and efficacy (Felder et al., 1995), and it has become a useful tool molecule in cannabinoid-related pharmacology studies (Devane et al., 1992). Cannabidiol (CBD, Fig. 1) is the second prevalent active ingredient in the Cannabis sativa L. but lacks detectable psychoactivity (Blessing et al., 2015). Although CBD interacts with CB1 and CB2 in vitro, it acts as an antagonist and an antidote to Δ9-THC, providing protection against certain negative effects caused by THC abuse (Hampson et al., 1998; Rong et al., 2017). Sativex (GW Pharmaceuticals, UK), an oromucosal spray with THC and CBD in a 1:1 ratio, has now received marketing authorization in Europe and Canada for the treatment for adult multiple sclerosis-related spasticity (Bifulco and Pisanti, 2015; Karschner et al., 2011; Wright et al., 2012).
Nowadays, the concept of phytocannabinoid is no longer limited to active ingredients from Cannabis sativa L., and a number of components have been identified in non-cannabis species, having important effects through the mediation of cannabinoid receptors (Gertsch et al., 2010; Sharma et al., 2015). For example, Amyrins, widely distributed in nature, are extremely potent on CB1 (Ki = 0.133 nM) over CB2 (Ki = 1,989 nM) (da Silva et al., 2011). Such naturally occurring molecules not only enrich the inventory of cannabinoids, but also offer potential leads for drug design. Cannabinoid-related drug development in return helps people understand the underlying mechanisms of how medicines act on human bodies (Hanus et al., 2016).
Multiple reports have revealed adverse health effects of synthetic cannabinoids with emphasis on psychosis-like effects. CB1 antagonism is considered as a promising therapeutic approach to treat overweight/obesity and related cardiometabolic disorders (Janero and Makriyannis, 2009). CB1-selective antagonists, SR141716A and its close analogs (MK-0364 and CP945,598), were first used for the treatment of obesity but later found to cause serious depression and anxiety (Di Marzo and Despres, 2009; Proietto et al., 2010; Traynor, 2007). Thus, clinical applications of these drugs were discontinued. How to minimize and overcome side effects is a challenge. It drives us to continuously explore the natural source to seek new active ingredients as templates to develop drugs with no or minor negative effects, for the sake of human health.
In this work, 82 compounds derived from 10 species (5 plants and 5 fungi) were screened on CB1 and CB2 receptors. These species grow in west China and have been reported to have beneficial effects to mental health and immune system (Table 1), but whether the cannabinoid receptors are engaged in their effects remains not known. A label-free dynamic mass redistribution (DMR) technology was applied that measured cellular responses to compounds by a resonant waveguide grating optical biosensor (Fang, 2011; Schroder et al., 2011). Active ingredients were found in three of the ten species. Among the 82 compounds, 4 compounds distributed in the Ganoderma species were identified to be active on the cannabinoid receptors, including three antagonists and a special agonist that may stimulate CB1 via a different mechanism. EC50 values of these compounds were between 20 and 35 μM, making them promising leads for drug design. Another three compounds were identified in the Lepidium meyenii Walp. (Brassicaceae) plant, effects of which were mediated through mechanisms much related to the signaling transduction pathways.
Section snippets
Materials
CP55,940 and U73122 were purchased from Sigma-Aldrich (St. Louis, MO), WIN55,212–2 from Absin Bioscience Inc. (Shanghai, China), and SR141716A and SR144528 from Tocris Biosciences (Ellisville, MO). Pertussis toxin was purchased from APExBio (Houston, TX), cholera toxin from Shanghai Yuan Ye Biotechnology Co., Ltd. (Shanghai, China), and LY294002 from Selleck Chemicals (Houston, TX). Cyclic monophosphate (cAMP)-Gi kit was purchased from cisbio (Number 62AM9PEB, Bedford, MA) for cAMP HTRF
Characterization of transfected CHO-CB1 and CHO-CB2 cell lines
DMR detections were performed with the transfected CHO-CB1 and CHO-CB2 cell lines so as to verify the transfection quality and characterize the models with specific tool molecules, and meanwhile to prove the suitability of applying the DMR technology on the cannabinoid receptors. Four high-affinity, commonly-used probe molecules were used in this work to fulfill the aims (Fig. 1).
CP55,940 is a non-selective agonist of CB1 and CB2. Reported Ki values, obtained from radiolabeling assays, are in a
Binding characteristics of CP55,940 and WIN55,212-2
During the model characterization, CP55,940 and WIN55,212–2 were used as agonist tool molecules, and some points should not be missed. First, DMR assay results showed that CP55,940 was non-selective between receptor subtypes and the transfection level of CB1 was higher than CB2. WIN55,212–2 was determined to be selective to CB2 over CB1 with EC50 differed by a factor of (CB1/CB2: 2,006/140.7 = ) 14.7. This result agrees with earlier reports (Felder et al., 1995; McPartland et al., 2007).
Second,
Conclusions
For decades, phytocannabinoids were considered as cannabinoids that occur naturally in the Cannabis sativa L. plant. Then more and more naturally occurring compounds distributed in species besides Cannabis sativa L. have been found active on the cannabinoid receptors. In this work, natural cannabinoids were identified in Lepidium meyenii Walp., and also in two species of Ganoderma (Ganoderma hainanense J.D. Zhao, L.W. Hsu & X.Q. Zhang and Ganoderma cochlear (Blume & T. Nees) Bres., Hedwigia).
Declaration of interest
None. The authors declare no competing financial interest.
Author contributions
Han Zhou and Xingrong Peng: provided equal contribution to this work. Han Zhou: conceived the present idea and designed the project; designed the models and performed the experiments; wrote the manuscript with input from all authors. Xingrong Peng: prepared the compound samples and provided information of the plants and fungi. Tao Hou: designed the models and performed the experiments. Nan Zhao: drafted the chemical structures and analyzed the data. Minghua Qiu: prepared the compound samples
Acknowledgement
This work was supported by the “State Key Program of National Natural Science of China (Grant No. U1508221)”, “Project of National Science Foundation of China (81803706)”, “Innovation Program of Science and Research” from DICP, CAS (DICP TMSR201601), and the funding for the construction of DICP-CMC Innovation Institute of Medicine. We thank Dr. Olivier Civelli at University of California, Irvine for generously providing plasmids for cell transfections.
References (91)
- et al.
CB2: therapeutic target-in-waiting
Prog. Neuro-Psychopharmacol. Biol. Psychiatry
(2012) - et al.
Wild and commercial mushrooms as source of nutrients and nutraceuticals
Food Chem. Toxicol.
(2008) - et al.
Cannabidiol as a potential treatment for anxiety disorders
Neurotherapeutics
(2015) - et al.
The endocannabinoid system and its therapeutic exploitation in multiple sclerosis: clues for other neuroinflammatory diseases
Prog. Neurobiol.
(2018) - et al.
Coffee with cinnamon - impact of phytochemicals interactions on antioxidant and anti-inflammatory in vitro activity
Food Chem.
(2014) - et al.
Phytocannabinoids: a unified critical inventory
Nat. Prod. Rep.
(2016) The cannabinoid receptors
Prostaglandins Other Lipid Mediat.
(2002)- et al.
Regulation of the expression of GABAA receptor subunits by an antiepileptic drug QYS
Neurosci. Lett.
(2006) - et al.
Administration of 2-arachidonoylglycerol ameliorates both acute and chronic experimental autoimmune encephalomyelitis
Brain Res.
(2011) - et al.
Interactions between cannabinoid receptor agonists and mu opioid receptor agonists in rhesus monkeys discriminating fentanyl
Eur. J. Pharmacol.
(2016)
A mushroom fruiting body-inducing substance inhibits activities of replicative DNA polymerases
Biochem. Biophys. Res. Commun.
Neuroprotective effects of the synthetic cannabinoid HU-210 in primary cortical neurons are mediated by phosphatidylinositol 3-kinase/AKT signaling
Mol. Cell. Neurosci.
The role of the CB1 receptor in the regulation of sleep
Prog. Neuro-Psychopharmacol. Biol. Psychiatry
GPR35 is a novel lysophosphatidic acid receptor
Biochem. Biophys. Res. Commun.
Phytochemical overview and medicinal importance of Coffea species from the past until now
Asian Pac. J. Trop. Med.
Protective effects of triterpenoids from Ganoderma resinaceum on H(2)O(2)-induced toxicity in HepG2 cells
Food Chem.
Unusual prenylated phenols with antioxidant activities from Ganoderma cochlear
Food Chem.
Lanostane triterpenoids from Ganoderma hainanense J. D. Zhao
Phytochemistry
Racemic meroterpenoids from Ganoderma cochlear
Fitoterapia
Antioxidant potential of low-grade coffee beans
Food Res. Int.
Sr141716a, a potent and selective antagonist of the brain cannabinoid receptor
FEBS Lett.
Cannabidiol in medical marijuana: research vistas and potential opportunities
Pharmacol. Res. : Off. J. Italian Pharmacol. Soc.
Natural cannabinoids: templates for drug discovery
Life Sci.
Hydantoin and thioamide analogues from Lepidium meyenii
Phytochem. Lett.
Macamides and their synthetic analogs: evaluation of in vitro FAAH inhibition
Bioorg. Med. Chem.
Ganoderma lucidum inhibits tumour cell proliferation and induces tumour cell death
Enzym. Microb. Technol.
Pharmacological effects of natural Ganoderma and its extracts on neurological diseases: a comprehensive review
Int. J. Biol. Macromol.
GPR55 and GPR35 and their relationship to cannabinoid and lysophospholipid receptors
Life Sci.
Towards a better understanding of medicinal uses of Carthamus tinctorius L. in traditional Chinese medicine: a phytochemical and pharmacological review
J. Ethnopharmacol.
Antiplasmodial lanostanes from the Ganoderma lucidum mushroom
J. Nat. Prod.
Functional selectivity in CB2 cannabinoid receptor signaling and regulation: implications for the therapeutic potential of CB2 ligands
Mol. Pharmacol.
The molecular and structural basis of advanced antiviral therapy for hepatitis C virus infection
Nat. Rev. Microbiol.
Medicinal use of cannabis in Europe: the fact that more countries legalize the medicinal use of cannabis should not become an argument for unfettered and uncontrolled use
EMBO Rep.
The effects of Delta(9)-tetrahydrocannabinol on the dopamine system
Nature
Multitarget drug discovery and polypharmacology
ChemMedChem
Potential anxiolytic- and antidepressant-like effects of salvinorin A, the main active ingredient of Salvia divinorum, in rodents
Br. J. Pharmacol.
Beneficial effects of Lepidium meyenii (Maca) on psychological symptoms and measures of sexual dysfunction in postmenopausal women are not related to estrogen or androgen content
Menopause
Dynamic mass redistribution as a means to measure and differentiate signaling via opioid and cannabinoid receptors
Assay Drug Dev. Technol.
Activation of cannabinoid receptors by the pentacyclic triterpene alpha,beta-amyrin inhibits inflammatory and neuropathic persistent pain in mice
Pain
Rituals, ceremonies and customs related to sacred trees with a special reference to the Middle East
J. Ethnobiol. Ethnomed.
HTRF: a technology tailored for drug discovery - a review of theoretical aspects and recent applications
Curr. Chem. Genom.
Medical uses of Carthamus tinctorius L. (Safflower): a comprehensive review from traditional medicine to modern medicine
Electron. Physician
A novel probe for the cannabinoid receptor
J. Med. Chem.
Cannabinoid CB2 receptor potentiates obesity-associated inflammation, insulin resistance and hepatic steatosis
PLoS One
CB1 antagonists for obesity--what lessons have we learned from rimonabant?
Nat. Rev. Endocrinol.
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Co-first authors: Han Zhou and Xingrong Peng provided equal contribution to this work.