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

SIMPLE SUMMARY: Kynurenine-3-monooxygenase (KMO) is an enzyme in the neurotoxic branch of the kynurenine pathway (KP) and is a target of inhibitors with therapeutic potential against neuroinflammatory and neurodegenerative diseases. Phytochemicals from hemp (Cannabis sativa L.) including phytocannabinoids and flavonoids, can modulate enzymes involved in the KP metabolism, but their direct inhibition on KMO is unknown. In the current study, we purified a unique C. sativa flavonoid, namely, cannflavin A (CFA), from the hemp aerial material at a gram-scale. We evaluated the anti-KMO activity of CFA and a panel of phytocannabinoids. CFA was the most active compound with an IC(50) of 29.4 μM, which was comparable to the positive control Ro 61-8048 (IC(50) = 5.1 μM). We used a modeling method to demonstrate the interactions between CFA and the KMO protein and measured the binding affinity by a biophysical tool (surface plasmon resonance; SPR). We also conducted a competitive SPR assay to show that CFA and Ro 61-8048 may bind to the same location of the KMO protein. Our findings help the understanding of C. sativa compounds’ effects on KMO. 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 (IC(50) = 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 [Formula: see text]. 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.