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

Abstract

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 μM, 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 4.1×10−5 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.