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 . 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.

Identification of potent inhibitors of kynurenine-3-monooxygenase from natural products: In silico and in vitro approaches

“…We performed the inhibition assay of KMOusingthe method described by Puopolo et al [ 22 ] with a slight modification, using Ro 61–8048 as the positive control. The test molecules were dissolved in 100 mM DMSO and diluted in assay buffer to 1 mM.…”

Investigating cannabinoids as P2X purinoreceptor 4 ligands by using surface plasmon resonance and computational docking

“…Recombinant human P2X4 (500 μg/mL stock solution in Dnase free water) was diluted to 40 μg/mL in sodium acetate 4.0. The protein was immobilized on a CM5 chip based on an amine coupling method as previously described with minor modification [ 19 ]. Briefly, the immobilization method involved five phases.…”

Chemistry and Biological Activities of Cannflavins of the Cannabis Plant

“…The cannflavin A-rich fraction was purified by dissolving in hexanes and filtration, followed by crystallization from EtOAc:acetone mixture (5:1) at 4°C to yield 38.7 g of cannflavin A. 34 …”

“…For example, Puopolo et al used 300 kg of dried hemp powder to obtain only 38.73 g of cannflavin A. 34 …”

“… 26 In another study, cannflavin A showed moderate antileishmanial and antitrypanosomal activities with IC 50 values of 14.6±3.7 and 1.9±0.8 μg/mL, respectively. 34 Cannflavin A also has promising docking energy against Leishmania major pteridine reductase ( E dock =−144.0 kJ/mol), comparable to methotrexate, the co-crystallized ligand ( E dock =−149.8 kJ/mol). 65 …”