Development of a miniaturized 384-well high throughput screen for the detection of substrates of cytochrome P-450 2D6 and 3A4 metabolism

The identification of a large number of biologically active chemical entiti es 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 c ytochrome P-450 (CYP) enzymes to evaluate the potential drug-drug interacti ons. CYP2D6 and CYP3A4 comprise the main human CYP enzymes involved in drug metabolism. The recent availability of specific CYP cDNA expression system s and the development of specific fluorescent probes have accelerated the a bility to develop robust in vitro assays in HTS format. The aim of this stu dy was to optimize conditions for the CYP2D6 and CYP3A4 HTS assays and subs equently adapt those assays to a miniaturized 384-well format. Assay conver sion to a miniaturized format presents certain difficulties, such as robust ness of the signal and of compound delivery. Thus the assay optimization in volved the comparison of different substrates to identify those most suitab le for use in a miniaturized format. Because of current technical limitatio ns in liquid dispensing of nanoliter volumes, assay sensitivity to organic solvents also provides a main concern during assay miniaturization. Therefo re, compound activity from redissolved dry films and from DMSO stocks direc tly delivered into assay buffer was compared. The data indicate that compou nd activity was comparable in both formats. The data support the conclusion that CYP2D6 and CYP3A4 in vitro metabolism assays can be successfully perf ormed in 384-well plate format and the substrate potencies, as evaluated by the IC50 values, determined.