Development of a Miniaturized 384-Well High Throughput Screen for the Detection of Substrates of Cytochrome P450 2D6 and 3A4 Metabolism

Abstract

The identification of a large number of biologically active chemical entities during high throughput screening (HTS) necessitates the incorporation of new strategies to identify compounds with druglike properties early during the lead prioritization and development process. One of the major steps in lead prioritization is the assessment of drug metabolism mediated by the cytochrome P450 (CYP) enzymes to evaluate the potential drug-drug interactions. CYP2D6 and CYP3A4 comprise the main human CYP enzymes involved in drug metabolism. The recent availability of specific CYP cDNA expression systems and the development of specific fluorescent probes have accelerated the ability to develop robust in vitro assays in HTS format. The aim of this study was to optimize conditions for the CYP2D6 and CYP3A4 HTS assays and subsequently adapt those assays to a miniaturized 384-well format. Assay conversion to a miniaturized format presents certain difficulties, such as robustness of the signal and of compound delivery. Thus the assay optimization involved the comparison of different substrates to identify those most suitable for use in a miniaturized format. Because of current technical limitations in liquid dispensing of nanoliter volumes, assay sensitivity to organic solvents also provides a main concern during assay miniaturization. Therefore, compound activity from redissolved dry films and from DMSO stocks directly delivered into assay buffer was compared. The data indicate that compound activity was comparable in both formats. The data support the conclusion that CYP2D6 and CYP3A4 in vitro metabolism assays can be successfully performed in 384-well plate format and the substrate potencies, as evaluated by the IC50 values, determined.

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

“…[27][28][29] Alternatively, to minimize the negative effect of DMSO on assay performance, test compounds could be delivered as redissolved dry films, according to recent data demonstrating comparable activity for compounds delivered in DMSO stocks or as dried compound films. 23 The latter approach may be preferable for further development of fully automated miniaturized assays and robotic applications.…”

“…[20][21][22] In addition, fluorometric assays in a 384-well format for the CYP3A4 and CYP2D6 isozymes have also been described. 23 However, many undesirable features of the generic (unmodified) fluorescent molecules, such as poor aqueous solubility, high background fluorescence (e.g., the fluorescence of unmetabolized substrate), and low signal-to-background ratios, limit their applications for low-volume ultra-high-throughput formats. Recently, highly miniaturized formats for primary screening assays involving several important pharmaceutical targets were developed.…”

Monitoring Compound Integrity With Cytochrome P450 Assays and qHTS

“…These enzymes have evolved to act as molecular incinerators in the liver and are known to interact with a wide range of substrates, making CYP assays ideal tools for quality assessment of diverse sets of compounds. In this study, we employed CYP 1A2 and 2D6 isozymes because we have previously confirmed that these assays perform well in 1536-well microplate formats, show low sensitivity to DMSO, 11,12 and are inhibited by a large number of compounds in typical compound collections (see PubChem AIDs 410 and 891, respectively). We examined compound storage in 1536-well microplates because this is central to our screening platform.…”

Luciferin IPA–Based Higher Throughput Human Hepatocyte Screening Assays for CYP3A4 Inhibition and Induction

“…the assays reported here should complement the current higher throughput approaches for the evaluation of P450-related drugdrug interactions. [16][17][18]…”