Inhibitory effects and oxidation of 6-methylcoumarin, 7-methylcoumarin and 7-formylcoumarinviahuman CYP2A6 and its mouse and pig orthologous enzymes

Abstract

1. Information about the metabolism of compounds is essential in drug discovery and development, risk assessment of chemicals and further development of predictive methods. 2. In vitro and in silico methods were applied to evaluate the metabolic and inhibitory properties of 6-methylcoumarin, 7-methylcoumarin and 7-formylcoumarin with human CYP2A6, mouse CYP2A5 and pig CYP2A19. 3. 6-Methylcoumarin was oxidized to fluorescent 7-hydroxy-6-methylcoumarin by CYP2A6 (Km: 0.64-0.91 µM; Vmax: 0.81-0.89 min(-1)) and by CYP2A5 and CYP2A19. The reaction was almost completely inhibited at 10 µM 7-methylcoumarin in liver microsomes of human and mouse, but in pig only 40% inhibition was obtained with the anti-CYP2A5 antibody or with methoxsalen and pilocarpine. 7-Methylcoumarin was a mechanism-based inhibitor for CYP2A6, but not for the mouse and pig enzymes. 7-Formylcoumarin was a mechanism-based inhibitor for CYP2As of all species. 4. Docking and molecular dynamics simulations of 6-methylcoumarin and 7-methylcoumarin in the active sites of CYP2A6 and CYP2A5 demonstrated a favorable orientation of the 7-position of 6-methylcoumarin towards the heme moiety. Several orientations of 7-methylcoumarin were possible in CYP2A6 and CYP2A5. 5. These results indicate that the active site of CYP2A6 has unique interaction properties for ligands and differs in this respect from CYP2A5 and CYP2A19.

Suitability of MMGBSA for the selection of correct ligand binding modes from docking results

“…Altogether, the stability, the distance of the closest carbon to the heme iron, and the binding energy estimations suggested that the primary SOM in 6-methylcoumarin would be C7 in pose 1 (Tables 1 and 2). This result, where C7 is the preferred SOM, correlates with the experimental results, which show that the primary metabolite is hydroxylated at C7 (Juvonen et al, 2016).…”

“…For CYP2A6, two known substrates, coumarin and 6-methylcoumarin, were selected. Both compounds are 7-hydroxylated in CYP2A6 oxidation (Juvonen et al, 2016;Pelkonen, Rautio, Raunio, & Pasanen, 2000). For AR, ligands were separated from the chosen protein crystal structures.…”

“…Specifically, we have studied the stability of both formed complexes and binding energies using MD simulation snapshots without and with energy minimization. Three protein targets that we have studied also previously (Juvonen et al, 2016;Kurkinen et al, 2018;Niinivehmas et al, 2011;Shubina, Niinivehmas, & Pentikäinen, 2015;Virtanen & Pentikäinen, 2010)-cytochrome P450 type 2A6 (CYP2A6), androgen receptor (AR), and phosphodiesterase 4B (PDE4B)-were utilized to evaluate the performance of MMGBSA in identifying ligand binding modes. CYP2A6 has a relatively small binding site for ligands, and thus, it is an important player in the oxidation of the smallest xenobiotics (Raunio & Rahnasto-Rilla, 2012).…”

Use of human liver and EpiSkin™ S9 subcellular fractions as a screening assays to compare the in vitro hepatic and dermal metabolism of 47 cosmetics‐relevant chemicals

“…The five chemicals for which the metabolites did not correlate with reported information were: 6‐methylcoumarin, dimethyl phthalate; nitrobenzene; geraniol and Basic Red 76. For 6‐methylcoumarin, the literature value was for liver microsomes supplemented with NADPH (Juvonen et al, ), which only represents Phase 1 reactions and would not produce the glucuronide detected in these S9 incubations. Although dimethyl phthalate was rapidly metabolized by both liver and EpiSkin S9, there were no metabolites detected, possibly due to the analytical conditions being optimal for the parent and not the metabolites.…”

“…Published information on the in vivo and/or in vitro metabolism was available for 35 of the chemicals tested, which was used to assess whether the liver and/or EpiSkin S9 produced relevant metabolites ( (Juvonen et al, 2016), which only represents Phase 1 reactions and would not produce the glucuronide detected in these S9 incubations.…”