Bryan D. Marks

Highly Miniaturized Formats for In Vitro Drug Metabolism Assays Using Vivid ® Fluorescent Substrates and Recombinant Human Cytochrome P450 Enzymes

Highly miniaturized P450 screening assays designed to enable facile analysis of P450 drug interactions in a 1536-well plate format with the principal human cytochrome P450 enzymes (CYP3A4, 2D6, 2C9, 2C19, and 1A2) and Vivid® fluorogenic substrates were developed. The detailed characterization of the assays included stability, homogeneity, and reproducibility of the recombinant P450 enzymes and the kinetic parameters of their reactions with Vivid® fluorogenic substrates, with a focus on the specific characteristics of each component that enable screening in a low-volume 1536-well plate assay format. The screening assays were applied for the assessment of individual cytochrome P450 inhibition profiles with a panel of selected assay modifiers, including isozyme-specific substrates and inhibitors. IC50 values obtained for the modifiers in 96- and 1536-well plate formats were similar and comparable with values obtained in assays with conventional substrates. An overall examination of the 1536-well assay statistics, such as signal-to-background ratio and Z′ factor, demonstrated that these assays are a robust, successful, and reliable tool to screen for cytochrome P450 metabolism and inhibition in an ultra-high-throughput screening format. (Journal of Biomolecular Screening 2005:56-66)

A Simultaneous Assessment of CYP3A4 Metabolism and Induction in the DPX-2 Cell Line

The DPX-2 cell line, a derivative of HepG2 cells, harbors human PXR and a luciferase-linked CYP3A4 promoter. These cells were used in a panel of cell-based assays for a parallel assessment of CYP3A4 induction, metabolism, and inhibition at the cellular level. CYP3A4 induction in the DPX-2 cell line by various agents was monitored in 96-well plates by a luciferase-based transcriptional activation assay. Of the prototypical CYP3A4 inducers examined, all exhibited elevated luciferase activity in DPX-2 cells. CYP3A4 enzyme activity in noninduced and rifampicin-induced DPX-2 cells was also assessed using Vivid fluorogenic substrates. Significantly elevated CYP3A4 activity levels (2.8-fold ±0.2-fold above DMSO-treated cells) were found in DPX-2 cells after 48 hours of exposure to rifampicin, but were unde-tectable in parental HepG2 cells. Rifampicin-induced activity levels were found to be suitable for assessing the inhibitory potential of new chemical entities in downstream CYP3A4 inhibition assays. The elevated CYP3A4 activity was inhibited 85% by 10 μM ketoconazole. In addition, a cytotoxicity assay to correct for possible toxic effects of compounds at the cellular level was applied. The comparative data obtained with a combination of the above assays suggests that the application of several independent in vitro technologies used in DPX-2 cells is the best possible strategy for the assessment of the complex phenomena of CYP3A4 induction and inhibition.

Development of the Predictor hERG Fluorescence Polarization Assay Using a Membrane Protein Enrichment Approach

The life-threatening consequences of acquired, or drug-induced, long QT syndrome due to block of the human ether-a-go-go-related gene (hERG) channel are well appreciated and have been the cause of several drugs being removed from the market in recent years because of patient death. In the last decade, the propensity for block of the hERG channel by a diverse and expanding set of compounds has led to the requirement that all new drugs be tested for hERG channel block in a functional patch-clamp assay. Because of the need to identify potential hERG blockers early in the discovery process, radiometric hERG binding assays are preferred over patch-clamp assays for compound triage, because of relative advantages in speed and cost. Even so, these radiometric binding assays are laborious and require dedicated instrumentation and infrastructure to cope with the regulatory and safety issues associated with the use of radiation. To overcome these limitations, we developed a homogeneous, fluorescence polarization-based assay to identify and characterize the affinity of small molecules for the hERG channel and have demonstrated tight correlation with data obtained from either radioligand binding or patch-clamp assays. Key to the development of this assay was a cell line that expressed highly elevated levels of hERG protein, which was generated by coupling expression of the hERG channel to that of a selectable cell surface marker. A high-expressing clone was isolated by flow cytometry and used to generate membrane preparations that contained >50-fold the typical density of hERG channels measured by [(3)H]astemizole binding. This strategy enabled the Predictor (Invitrogen, Carlsbad, CA) hERG fluorescence polarization assay and should be useful in the development of other fluorescence polarization-based assays that use membrane proteins.

High-Throughput screeening assays for CYP2B6 metabolism and inhibition usuing fluorogenic vivid substrates

CYP2B6 is a highly polymorphic P450 isozyme involved in the metabolism of endo-and xenobiotics with known implications for the activation of many procarcinogens resulting in carcinogenesis. However, lack of validated high-throughput screening (HTS) CYP2B6 assays has limited the current understanding and full characterization of this isozyme’s involvement in human drug metabolism. Here, we have developed and characterized a fluorescence-based HTS assay employing recombinant human CYP2B6 and 2 novel fluorogenic substrates (the Vivid CYP2B6 Blue and Cyan Substrates). Assay validation included testing the inhibitory potency of a panel of drugs and compounds known to be metabolized by this isozyme, including CYP2B6 substrates, inhibitors, and known inducers. Compound rankings based on inhibitory potency in the Vivid CYP2B6 Blue and Cyan Assays matched compound rankings based on relative affinity measurements from previously published data (Ki, Kd, or Km values) for the CYP2B6 isozyme. In conclusion, these assays are proven to be robust and sensitive, with broad dynamic ranges and kinetic parameters allowing screening in HTS mode of a large panel of compounds for CYP2B6 metabolism and inhibition, and are a valuable new tool for CYP2B6 studies.

High-throughput screening assays for the assessment of CYP2C9*1, CYP2C9*2, and CYP2C9*3 metabolism using fluorogenic Vivid substrates.

CYP2C9 is a genetically polymorphic human cytochrome P450 isozyme involved in the oxidative metabolism of many drugs, including nonsteroidal anti-inflammatory compounds. Individuals genotyped heterozygous or homozygous for CYP2C9 allelic variants have demonstrated altered metabolism of some drugs primarily metabolized by CYP2C9. The ability to expand screening of CYP2C9 allelic variants to a larger set of drugs and pharmaceutical agents would contribute to a better understanding of the significance of CYP2C9 polymorphisms in the population and to predictions of possible outcomes. The authors report the development of an in vitro fluorescence-based assay employing recombinant CYP2C9 variants (CYP2C9*1, CYP2C9*2, and CYP2C9*3) and fluorogenic Vivid® CYP2C9 substrates to explore the effects of CYP2C9 polymorphisms on drug metabolism, using drugs primarily metabolized by CYP2C9. Several chemically diverse fluorogenic substrates (Vivid® CYP2C9 blue, green, and red substrates) were used as prototypic probes to obtain in vitro CYP2C9 metabolic rates and kinetic parameters, such as apparent Km, Vmax, and Vmax/Km ratios for each allelic variant. In addition, a diverse panel of drugs was screened as assay modifiers with CYP2C9*1, CYP2C9*2, CYP2C9*3, and the fluorogenic Vivid® CYP2C9 substrates. The inhibitory potential of this large group of chemically diverse drugs and compounds has been assessed on the basis of their ability to compete with Vivid® CYP2C9 substrates in fluorescent reporter assays, thus providing a sensitive and quick assessment of polymorphism-dependent changes in CYP2C9 metabolism.