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.
References
1.
Gordon EM, Gallop MA, Campbell D, et al.Combinatorial organic synthesis: application to drug discovery. Eur J Med Chem1995;30:S237-S248.
2.
Oldenburg KR. Current and future trends in high throughput screening for drug discovery. Annu Rep Med Chem1998;33:301-311.
Krebs R. Novel technology to improve pharmaceutical productivity. Curr Opin Drug Discov Dev1999;2:239-243.
5.
Rodrigues AD. Rational high-throughput screening in preclinical drug metabolism. Med Chem Res1998;8:422-433.
6.
Smith DA, van de Waterbeemd H.Pharmacokinetics and metabolism in early drug discovery. Curr Opin Chem Biol1999;3:373-378.
7.
Tarbit MH, Berman J. High-throughput approaches for evaluating absorption, distribution, metabolism and excretion properties of lead. Curr Opin Chem Biol1998;2:411-416.
8.
Venkatesh S, Lipper RA. Role of the development scientist in compound lead selection and optimization. J Pharm Sci1999;89:145-154.
9.
Lin JH, Lu AY. Inhibition and induction of cytochrome P450 and the clinical implications. Clin Parmacokinet1998;35:361-390.
10.
Maffia AM, Kariv I, Oldenburg KR. Miniaturization of a mammalian cell-based assay: luciferase reporter gene readout in 3,uL 1536-well format. J Biomol Screen1999;4:137-141.
11.
Pin SS, Kariv I, Graciani NR, et al.Analysis of protein-peptide interaction by a miniaturized fluorescence polarization assay using cyclin dependent kinase 2/cyclin E as a model systemAnal Biochem1999;275:156-161.
12.
Schmider J, von Moltke LL, Shader RI, et al.Extrapolation in vitro data on drug metabolism to in vivo pharmacokinetics: valuation of the pharmacokinetic interaction between amitriptyline and fluoxetine. Drug Metab Rev1999;31:545-560.
13.
White RE. High-throughput screening in drug metabolism and pharmacokinetic support of drug discovery. Annu Rei Pharinacol Toxicol2000;40:133-157.
14.
Crespi CL, Miller VP. Penman BW. High throughput screening for inhibition of cytochrome P450 metabolism. Med Cheem Res1998;8:457-471.
15.
Parkinson A. An overview of current cytochrome P450 technology for assessing the safety and efficacy of new materials. Toxicology1996;24:45-57.
16.
Spatzenegger M, Jaeger W. Clinical importance of hepatic cytochrome P450 in drug metabolism. Drutg Metab Rev1995;27:397-417.
17.
Rauschenback R, Gieschen H, Salomon B, et al.Development of a V79 cell line expressing human cytochrome P450 2D6 and its application as a metabolic screening tool. Environ Toxicol Pharmacol1997;3:31-39.
18.
Knodell RG, Browne DG, Gwozdz GP., et al.Differential inhibition of individual human liver cytochromes P450 by cimetidine. Gastroenterology1991;101:1680-1691.
19.
Ayrton J, Plumb R, Leavens WJ, et al.Application of a generic fast gradient liquid chromatography tandem mass spectrometry method for the analysis of cytochrome P450 probe substrates. Rapid Commun Mass Spectrom1998X12:217-224.
20.
Yin H, Racha J, Li SY, et al.Automated high throughput human CYP isoform activity assay using SPEC-LC/MS method: application in CYP inhibition evaluation. Xenobiotica2000;30:141-154.
21.
Moody GC, Griffin SJ, Mather AN, et al.Fully automated analysis of activities catalysed by the major human liver cytochrome P450 (CYP) enzymes: assessment of human CYP inhibition potential. Xenobiotica1999;29:53-75.
22.
Crespi CL, Miller VP, Penman BW. Microtiter plate assays for inhibition of human, drug-metabolizing cytochromes P450. Anal Biochem1997;248:188-190.
23.
Palamanda JR, Favreau L, Lin C-C.et al.Validation of a rapid microtiter plate assay to conduct cytochrome P450 2D6 enzyme inhibition studies. Drug Discov Today1998;3:466-470.
24.
Pope AJ, Haupts UM, Moore KJ. Homogeneous fluorescence read-outs for miniaturized high-throughput screening: theory and practice. Drug Discov Today;1999;4:350-362.
25.
Swartzman EE, Miraglia SJ, Michelotti JM, et al.A homogeneous and multiplexed immunoassay for high-throughput screening using fluorometric microvolume assay technology. Anal Biochein1999;271:143-151.
26.
Silverman L, Campbell R, Broach JR. New assay technologies for high-throughput screening. Curr Opin Chem Biol1998;2:397-403.
27.
Segel I. Enzvme Kinetics Behavior and Anal'ysis of Rapid Equilibrium and Steady State Enzyme Syistems. New York: John Wiley & Sons, 1993.
28.
Vielnascher E, Spatzenegger M, Mayrhofer A, et al.Metabolism of dextromethophan in human liver microsomes a rapid HPLC assay to monitor cytochrome P450 2D6 activity. Pharmazie1996;551:586-588.
29.
Zhang J-H, Chung TDY, Oldenburg KR. A simple statistical parameter for use in evaluation and validation of high throughput screening sssays. J Biomol Screening1999;4:67-73.
30.
Cashman JR, Young Z, Yang L, et al.Stereo- and regio-selective N- and S-oxidation of tertiary amines and sulfides in the presence of adult human liver microsomes. Drug Metab Dispos1993;21:492-501.
31.
Daly AK, Fairbrother KS, Smart J. Recent advances in understanding the molecular basis of polymorphisms in genes encoding cytochrome P450 enzymes. Toxicol Lett1998;102-103:143-147.
32.
Kurz H, Fichtl B. Binding of drugs to tissues. Drug Metab Rev1983;14:467-510.
33.
Pacifici GM, Viani A. Methods of determining plasma and tissue binding of drugs. Clin Pharmacokinet1992;23:449-468.
34.
Obach RS. Nonspecific binding to microsomes: impact on scale-up of in vitro intrinsic clearance to hepatic clearance as assessed through examination of warfarin, imipramine, and propranolol. Drug Metab Dispos1997;25:1359-1369.
