Inhibition of Recombinant Cytochrome P450 Isoforms 2D6 and 2C9 by Diverse Drug-like Molecules

Abstract

The affinities of a diverse set of 500 drug-like molecules to cytochrome P450 isoforms 2C9 and 2D6 were measured using recombinant expressed enzyme. The dose-response curve of each compound was fitted with a series of equations representing typical or various types of atypical kinetics. Atypical kinetics was identified where the Akaike Information Criterion, plus other criteria, suggested the kinetics was more complex than expected for a Michaelis-Menten model. Approximately 20% of the compounds were excluded due to poor solubility, and approximately 15% were excluded due to fluorescence interference. Of the remaining compounds, roughly half were observed to bind with an affinity of 200 microM or lower for each of the two isoforms. Atypical kinetics was observed in 18% of the compounds that bind to cytochrome 2C9, but less than 2% for 2D6. The resulting collection of competitive inhibitors and inactive compounds were analyzed for trends in binding affinity. For CYP2D6, a clear relationship between polar surface area and charge was observed, with the most potent inhibitors having a formal positive charge and a low percent polar surface area. For CYP2C9, no clear trend between activity and physicochemical properties could be seen for the group as a whole; however, certain classes of compounds have altered frequencies of activity and atypical kinetics.

The AmpliChip CYP450 test: cytochrome P450 2D6 genotype assessment and phenotype prediction

“…This could be explained by non-genetic factors having been shown to reduce CYP2D6 expression and/or activity. 17,18 On the other hand, limitations in phenotype measurement could also contribute to these discrepancies. Indeed, in contrast to the PM phenotype, where a DEM MR antimode can be precisely defined, the definition for the IM and UM phenotype is less clear.…”

A Numerical Method for Analysis of In Vitro Time-Dependent Inhibition Data. Part 1. Theoretical Considerations

“…Non-MM or atypical saturation kinetics occurs when an enzyme-substrate-substrate (ESS) complex is formed (Korzekwa et al, 2014b). CYP2C9 has been shown to display multisubstrate interactions approximately 20% of the time, whereas CYP2D6 was almost always competitive (McMasters et al, 2007). Although a similar study has not been reported for CYP3A4, the numerous reports of CYP3A4 non-MM kinetics suggest that multisubstrate interaction kinetics is common (Wrighton et al, 2000).…”

“…This results in some unusual kinetics, presumably due to simultaneous interaction of multiple substrates or inhibitors with the active site (Huang et al, 1981;Lasker et al, 1982;Atkins, 2005;McMasters et al, 2007). Non-MM or atypical saturation kinetics occurs when an enzyme-substrate-substrate (ESS) complex is formed (Korzekwa et al, 2014b).…”

“…For example, numerous studies have reported that two different substrates can simultaneously occupy the P450 active sites, resulting in partial inhibition, activation, or activation followed by inhibition (Shou et al, 1994;Korzekwa et al, 1998;Atkins, 2005;McMasters et al, 2007). After substrate addition in a TDI experiment, the effect of the inhibitor on substrate metabolism may not be competitive inhibition if an ESI complex can be formed.…”

A Numerical Method for Analysis of In Vitro Time-Dependent Inhibition Data. Part 2. Application to Experimental Data

“…Although none of the five TDIs here contains methylenedioxy groups, one such published dataset was evaluated, namely inhibition of CYP2D6 by methylenedioxymethamphetamine (Heydari et al, 2004). It was anticipated that this dataset would have an MM base because CYP2D6 is unlikely to display EII binding kinetics (McMasters et al, 2007). Analysis by the MM-only and MM-quasi-irreversible models is shown in Fig.…”