Fluorometric High‐Throughput Screening for Inhibitors of Cytochrome P450

Abstract

Rapid screening for cytochrome P450 inhibitors is part of the current paradigm for avoiding development of drugs likely to give clinical pharmacokinetic drug‐drug interactions and associated toxicities. We have developed microtiter plate‐based, direct, fluorometric assays for the activities of the principal human drug‐metabolizing enzymes, CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A4, as well as for CYP2A6, which is an important enzyme in environmental toxicology. These assays are rapid and compatible with existing high‐throughput assay instrumentation. For CYP1A2, CYP2C8, CYP2C9, CYP2C19, and CYP2D6, the potency of enzyme inhibition (IC50) is consistent regardless of the probe substrate or assay method employed. In contrast, CYP3A4 inhibition for an individual inhibitor shows significant differences in potency (>300‐fold) depending on the probe substrate being used. We have investigated these differences through the use of several structurally distinct fluorescent substrates for CYP3A4 and several classical substrate probes (e.g., testosterone, nifedipine, and midazolam), with a panel of known, clinically significant, CYP3A4 inhibitors. The use of multiple probe substrates appears to be needed to characterize the inhibition potential of xenobiotics for CYP3A4.

Defining Desirable Central Nervous System Drug Space through the Alignment of Molecular Properties, in Vitro ADME, and Safety Attributes

“…Data for the following in vitro safety end points were generated in house via HT assays: (a) drug-drug interactions, assessed via inhibition of cytochrome P450 (CYPs) monooxygenase enzymes, in particular, 3A4 and 2D6 (28); (b) hERG liability, assessed via inhibition of dofetilide binding (30) as a surrogate indicator of hERG potassium ion channel effects (blocking of hERG may result in prolongation of the QT interval of cardiac rhythm) (29), and (c) cellular toxicity, measured as activity in an in vitro cellular toxicity assay as a surrogate for acute in vivo toxicity (32). Assays were performed via reported methods as described previously for DDI (28) and dofetilide binding (30). The cellular toxicity data was generated using a transformed human liver epithelia cell line where ATP levels were detected using a bioluminescent end point.…”

High-Throughput Radiometric CYP2C19 Inhibition Assay Using Tritiated (S)-Mephenytoin

“…This requirement renders the procedures relatively laborious, time-consuming, and not ideally suited for screening the large number of compounds typically required in an industrial drug discovery setting. An alternative assay using the fluorogenic nonselective CYP2C19 substrate 3-cyano-7-ethoxycoumarin has been described (Miller et al, 2000). Even though fluorometric P450 assays are rapid, easy to perform, and amenable to automation, they suffer from a number of limitations, such as the absence of selective probes, the need to use recombinant enzyme rather than HLMs, imperfect correlation of IC 50 values with those determined using classic drug substrates (Cohen et al, 2003), and fluorescence interference by many test compounds.…”

In Vitro Metabolism and Inhibitory Effects of Pranlukast in Human Liver Microsomes

“…Effect of Pranlukast on Metabolism with Human BLymphoblastoid Cells Expressing Human CYPs Metabolism with human B-lymphoblastoid cells expressing human CYPs was measured according to methods of Miller et al 4) Pranlukast was added into reaction mixtures at a concentration of 1 or 10 mmol/l. The data are presented as the means of duplicate samples.…”