Inhibitory Effects of Cannabinoids on Acetylcholinesterase and Butyrylcholinesterase Enzyme Activities

Introduction: Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) are two cholinergic enzymes catalyzing the reaction of cleaving acetylcholine into acetate and choline at the neuromuscular junction. Abnormal hyperactivity of AChE and BChE can lead to cholinergic deficiency, which is associated with several neurological disorders including cognitive decline and memory impairments. Preclinical studies support that some cannabinoids including cannabidiol (CBD) and tetrahydrocannabinol (THC) may exert pharmacological effects on the cholinergic system, but it remains unclear whether cannabinoids can inhibit AChE and BChE activities. Herein, we aimed to evaluate the inhibitory effects of a panel of cannabinoids including CBD, Δ8-THC, cannabigerol (CBG), cannabigerolic acid (CBGA), cannabicitran (CBT), cannabidivarin (CBDV), cannabichromene (CBC), and cannabinol (CBN) on AChE and BChE activities. Methods: The inhibitory effects of cannabinoids on the activities of AChE and BChE enzymes were evaluated with the Ellman method using acetyl- and butyryl-thiocholines as substrates. The inhibition mechanism of cannabinoids on AChE and BChE was studied with enzyme kinetic assays including the Lineweaver-Burk and Michaelis-Menten analyses. In addition, computational-based molecular docking experiments were performed to explore the interactions between the cannabinoids and the enzyme proteins. Results: Cannabinoids including CBD, Δ8-THC, CBG, CBGA, CBT, CBDV, CBC, and CBN (at 200 µM) inhibited the activities of AChE and BChE by 70.8, 83.7, 92.9, 76.7, 66.0, 79.3, 13.7, and 30.5%, and by 86.8, 80.8, 93.2, 87.1, 77.0, 78.5, 27.9, and 22.0%, respectively. The inhibitory effects of these cannabinoids (with IC50 values ranging from 85.2 to \u3e200 µM for AChE and 107.1 to \u3e200 µM for BChE) were less potent as compared to the positive control galantamine (IC50 1.21 and 6.86 µM for AChE and BChE, respectively). In addition, CBD, as a representative cannabinoid, displayed a competitive type of inhibition on both AChE and BChE. Data from the molecular docking studies suggested that cannabinoids interacted with several amino acid residues on the enzyme proteins, which supported their overall inhibitory effects on AChE and BChE. Conclusion: Cannabinoids showed moderate inhibitory effects on the activities of AChE and BChE enzymes, which may contribute to their modulatory effects on the cholinergic system. Further studies using cell-based and in vivo models are warranted to evaluate whether cannabinoids’ neuroprotective effects are associated with their anti-cholinesterase activities

Identification of SARS-CoV-2 Main Protease Inhibitors from a Library of Minor Cannabinoids by Biochemical Inhibition Assay and Surface Plasmon Resonance Characterized Binding Affinity

The replication of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is mediated by its main protease (Mpro), which is a plausible therapeutic target for coronavirus disease 2019 (COVID-19). Although numerous in silico studies reported the potential inhibitory effects of natural products including cannabis and cannabinoids on SARS-CoV-2 Mpro, their anti-Mpro activities are not well validated by biological experimental data. Herein, a library of minor cannabinoids belonging to several chemotypes including tetrahydrocannabinols, cannabidiols, cannabigerols, cannabichromenes, cannabinodiols, cannabicyclols, cannabinols, and cannabitriols was evaluated for their anti-Mpro activity using a biochemical assay. Additionally, the binding affinities and molecular interactions between the active cannabinoids and the Mpro protein were studied by a biophysical technique (surface plasmon resonance; SPR) and molecular docking, respectively. Cannabinoids tetrahydrocannabutol and cannabigerolic acid were the most active Mpro inhibitors (IC50 = 3.62 and 14.40 μM, respectively) and cannabigerolic acid had a binding affinity KD=2.16×10−4 role= presentation \u3e=2.16×10−4 M). A preliminary structure and activity relationship study revealed that the anti-Mpro role= presentation \u3eMpro effects of cannabinoids were influenced by the decarboxylation of cannabinoids and the length of cannabinoids’ alkyl side chain. Findings from the biochemical, biophysical, and computational assays support the growing evidence of cannabinoids’ inhibitory effects on SARS-CoV-2 Mpro

Anti-Ferroptotic Effect of Cannabidiol in Human Skin Keratinocytes Characterized by Data-Independent Acquisition-Based Proteomics

Skin cells are susceptible to oxidative stress and various types of cell death, including an iron-dependent form known as ferroptosis. Cannabidiol (CBD) can protect skin cells against oxidative stress, but whether this is attributed to the inhibition of ferroptosis is unknown. Herein, we evaluated the anti-ferroptotic effect of CBD in human keratinocytes using biochemical assays (radical scavenging and iron chelating) and cell-based models (for lipid peroxidation and intracellular iron). CBD’s anti-ferroptotic effect was further characterized by proteomic analysis. This study identifies anti-ferroptosis as a mechanism of CBD’s skin protective effects

Gram-Scale Preparation of Cannflavin A from Hemp (Cannabis sativa L.) and Its Inhibitory Effect on Tryptophan Catabolism Enzyme Kynurenine-3-Monooxygenase

Inhibitors targeting kynurenine-3-monooxygenase (KMO), an enzyme in the neurotoxic kynurenine pathway (KP), are potential therapeutics for KP metabolites-mediated neuroinflammatory and neurodegenerative disorders. Although phytochemicals from Cannabis (C. sativa L.) have been reported to show modulating effects on enzymes involved in the KP metabolism, the inhibitory effects of C. sativa compounds, including phytocannabinoids and non-phytocannabinoids (i.e., cannflavin A; CFA), on KMO remain unknown. Herein, CFA (purified from hemp aerial material at a gram-scale) and a series of phytocannabinoids were evaluated for their anti-KMO activity. CFA showed the most active inhibitory effect on KMO, which was comparable to the positive control Ro 61-8048 (IC50 = 29.4 vs. 5.1 mu M, respectively). Furthermore, a molecular docking study depicted the molecular interactions between CFA and the KMO protein and a biophysical binding assay with surface plasmon resonance (SPR) technique revealed that CFA bound to the protein with a binding affinity of 4.1x10(-)(5) M. A competitive SPR binding analysis suggested that CFA and Ro 61-8048 bind to the KMO protein in a competitive manner. Our findings show that C. sativa derived phytochemicals, including CFA, are potential KMO inhibitors, which provides insight into the development of therapeutics targeting the KP and its related pathological conditions

Gram-Scale Preparation of Cannflavin A from Hemp (Cannabis sativa L.) and Its Inhibitory Effect on Tryptophan Catabolism Enzyme Kynurenine-3-Monooxygenase

Inhibitors targeting kynurenine-3-monooxygenase (KMO), an enzyme in the neurotoxic kynurenine pathway (KP), are potential therapeutics for KP metabolites-mediated neuroinflammatory and neurodegenerative disorders. Although phytochemicals from Cannabis (C. sativa L.) have been reported to show modulating effects on enzymes involved in the KP metabolism, the inhibitory effects of C. sativa compounds, including phytocannabinoids and non-phytocannabinoids (i.e., cannflavin A; CFA), on KMO remain unknown. Herein, CFA (purified from hemp aerial material at a gram-scale) and a series of phytocannabinoids were evaluated for their anti-KMO activity. CFA showed the most active inhibitory effect on KMO, which was comparable to the positive control Ro 61-8048 (IC50 = 29.4 vs. 5.1 mu M, respectively). Furthermore, a molecular docking study depicted the molecular interactions between CFA and the KMO protein and a biophysical binding assay with surface plasmon resonance (SPR) technique revealed that CFA bound to the protein with a binding affinity of 4.1x10(-)(5) M. A competitive SPR binding analysis suggested that CFA and Ro 61-8048 bind to the KMO protein in a competitive manner. Our findings show that C. sativa derived phytochemicals, including CFA, are potential KMO inhibitors, which provides insight into the development of therapeutics targeting the KP and its related pathological conditions