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)
Bugrim A, Nikolskaya T, Nikolsky Y: Early prediction of drug metabolism and toxicity: systems biology approach and modeling. Drug Discov Today2004;9:127-135.
2.
Kariv I, Rourick RA, Kassel DB, Chung TD: Improvement of “hit-to-lead” optimization by integration of in vitro HTS experimental models for early determination of pharmacokinetic properties. Comb Chem High Throughput Screen2002;5:459-472.
3.
Hemmila IA, Hurskainen P: Novel detection strategies for drug discovery. Drug Discov Today2002;7:S150-S156.
4.
Atterwill CK, Wing MG: In vitro preclinical lead optimisation technologies (PLOTs) in pharmaceutical development. Toxicol Lett2002;127:143-151.
5.
Tanaka E: Clinically important pharmacokinetic drug-drug interactions: role of cytochrome P450 enzymes. J Clin Pharm Ther1998;23:403-416.
6.
van de Waterbeemd H: High-throughput and in silico techniques in drug metabolism and pharmacokinetics. Curr Opin Drug Discov Devel2002;5:33-43.
7.
Masimirembwa CM, Thompson R, Andersson TB: In vitro high throughput screening of compounds for favorable metabolic properties in drug discovery. Comb Chem High Throughput Screen2001;4:245-263.
8.
Wolcke J, Ullmann D: Miniaturized HTS technologies—uHTS. Drug Discov Today2001;6:637-646.
9.
Moody GC, Griffin SJ, Mather AN, McGinnity DF, Riley RJ: 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.
10.
McGinnity DF, Parker AJ, Soars M, Riley RJ: Automated definition of the enzymology of drug oxidation by the major human drug metabolizing cytochrome P450s. Drug Metab Dispos2000;28:1327-1334.
11.
Draper AJ, Madan A, Latham J, Parkinson A: Development of a non-high pressure liquid chromatography assay to determine [14C]chlorzoxazone 6-hydroxylase (CYP2E1) activity in human liver microsomes. Drug Metab Dispos1998;26:305-312.
12.
Draper AJ, Madan A, Smith K, Parkinson A: Development of a non-high pressure liquid chromatography assay to determine testosterone hydroxylase (CYP3A) activity in human liver microsomes. Drug Metab Dispos1998;26:299-304.
13.
Kobayashi K, Yamamoto T, Taguchi M, Chiba K: High-performance liquid chromatography determination of N- and O-demethylase activities of chemicals in human liver microsomes: application of postcolumn fluorescence derivatization using Nash reagent. Anal Biochem2000;284:342-347.
14.
van’t Klooster GA, Woutersen-van Nijnanten FM, Kolker HJ, Noordhoek J, van Miert AS: Improved high-performance liquid chromatographic method for the determination of ethylmorphine and its metabolites in microsomal incubations and cell culture media. J Chromatogr1992;579:158-164.
15.
Chu I, Favreau L, Soares T, Lin C, Nomeir AA: Validation of higher-through put high-performance liquid chromatography/atmospheric pressure chemical ionization tandem mass spectrometry assays to conduct cytochrome P450s CYP2D6 and CYP3A4 enzyme inhibition studies in human liver microsomes. Rapid Commun Mass Spectrom2000;14:207-214.
16.
Rao MN, Biju B, Ansar AK, Mujeeb S, Ramesh M, Srinivas NR: “Open access” generic method for continuous determination of major human CYP450 probe substrates/metabolites and its application in drug metabolism studies. Xenobiotica2003;33:1233-1245.
17.
Cali JJ, Frackman S, Ho S, et al: Luciferin derivatives in luminescent assay formats for rapid screening of multiple compounds against P450 activities [poster 12020]. Paper presented at 9th Annual SBS Conference, Portland, OR, September 2003.
18.
Nakamura K, Hanna IH, Cai H, Nishimura Y, Williams KM, Guengerich FP: Coumarin substrates for cytochrome P450 2D6 fluorescence assays. Anal Biochem2001;292:280-286.
19.
Roser R, Thomas H: A direct, highly sensitive fluorometric assay for a microsomal cytochrome P450-mediated O-demethylation using a novel coumarin analog as substrate. Z Naturforsch [C]2000;55:915-922.
20.
Miller VP, Stresser DM, Blanchard AP, Turner S, Crespi CL: Fluorometric high-throughput screening for inhibitors of cytochrome P450. Ann N Y Acad Sci2000;919:26-32.
21.
Chauret N, Tremblay N, Lackman RL, et al: Description of a 96-well plate assay to measure cytochrome P4503A inhibition in human liver microsomes using a selective fluorescent probe. Anal Biochem1999;276:215-226.
22.
Bapiro TE, Egnell AC, Hasler JA, Masimirembwa CM: Application of higher throughput screening (HTS) inhibition assays to evaluate the interaction of antiparasitic drugs with cytochrome P450s. Drug Metab Dispos2001;29:30-35.
23.
Kariv I, Fereshteh MP, Oldenburg KR: Development of a miniaturized 384-well high throughput screen for the detection of substrates of cytochrome P450 2D6 and 3A4 metabolism. J Biomol Screen2001;6:91-99.
24.
Lavery P, Brown MJ, Pope AJ: Simple absorbance-based assays for ultra-high throughput screening. J Biomol Screen2001;6:3-9.
25.
Wu G, Irvine J, Luft C, Pressley D, Hodge CN, Janzen B: Assay development and high-throughput screening of caspases in microfluidic format. Comb Chem High Throughput Screen2003;6:303-312.
26.
Zlokarnik G, Volak LP, Andrew MA, et al: Use of fluorescent probes for detection of different types of CYP450-drug interactions [poster 49]. Paperpresented at DMW/ISSX meeting, St. Andrews, Scotland, June 2000.
27.
Marks BD, Smith RW, Braun HA, Ozers MS, Lebakken C, Trubetskoy OV: A novel HTS assay to screen for CYP2E1 metabolism and inhibition using a fluorogenic Vivid® P450 substrate. Assay Drug Dev Technol2002;1:73-81.
28.
Marks BD, Goossens TA, Braun HA, et al: High-throughput screening assays for CYP2B6 metabolism and inhibition using fluorogenic Vivid® substrates. AAPS Pharm Sci2003;5:E18.
29.
Marks BD, Thompson DV, Goossens TA, Trubetskoy OV: HTS assays for the assessment of CYP2C9*1, CYP2C9*2, and CYP2C9*3 metabolism using fluorogenic Vivid® substrates. J Biomol Screen2004;9:439-449.
30.
Zlokarnik G, Volak LP, Andrew MA, et al: Fluorogenic Vivid® substrates used to generate large data sets for SAR modeling of CYP3A4-drug interactions [poster 353]. Paper presented at 6th International ISSX Meeting, Munich, Germany, October 2001.
31.
Shaw PM, Hosea NA, Thompson DV, Lenius JM, Guengerich FP: Reconstitution premixes for assays using purified recombinant human cytochrome P450, NADPH-cytochrome P450 reductase, and cytochrome b5. Arch Biochem Biophys1997;348:107-115.
32.
Cohen LH, Remley MJ, Raunig D, Vaz AD: In vitro drug interactions of cytochrome p450: an evaluation of fluorogenic to conventional substrates. Drug Metab Dispos2003;31:1005-1015.
33.
Omura T, Sato R: The carbon monoxide-binding pigment of liver microsomes. J Biol Chem1964;239:2370-2385.
34.
Zhang JH, Chung TD, Oldenburg KR: A simple statistical parameter for use in evaluation and validation of high throughput screening assays. J Biomol Screen1999;4:67-73.
35.
Cho US, Ahn HJ, Park EY, Dong MS, Kim KH: Influence of ligand binding to human cytochrome P-450 1A2: conformational activation and stabilization by alpha-naphthoflavone. Biochim Biophys Acta2001;1546:412-421.
36.
Stresser DM, Blanchard AP, Turner SD, et al: Substrate-dependent modulation of CYP3A4 catalytic activity: analysis of 27 test compounds with four fluorometric substrates. Drug Metab Dispos2000;28:1440-1448.
37.
Venkatakrishnan K, von Moltke LL, Greenblatt DJ: Application of the relative activity factor approach in scaling from heterologously expressed cytochromes p450 to human liver microsomes: studies on amitriptyline as a model substrate. J Pharmacol Exp Ther2001;297:326-337.
38.
Venkatakrishnan K, Von Moltke LL, Greenblatt DJ: Human drug metabolism and the cytochromes P450: application and relevance of in vitro models. J Clin Pharmacol2001;41:1149-1179.
39.
Nakajima M, Tane K, Nakamura S, Shimada N, Yamazaki H, Yokoi T: Evaluation of approach to predict the contribution of multiple cytochrome P450s in drug metabolism using relative activity factor: effects of the differences in expression levels of NADPH-cytochrome P450 reductase and cytochrome b(5) in the expression system and the differences in the marker activities. J Pharm Sci2002;91:952-963.
40.
Crespi CL, Miller VP, Penman BW: Microtiter plate assays for inhibition of human, drug-metabolizing cytochromes P450. Anal Biochem1997;248:188-190.
41.
Easterbrook J, Lu C, Sakai Y, Li AP: Effects of organic solvents on the activities of cytochrome P450 isoforms, UDP-dependent glucuronyl transferase, and phenol sulfotransferase in human hepatocytes. Drug Metab Dispos2001;29:141-144.
42.
Egnell AC, Houston B, Boyer S: In vivo CYP3A4 heteroactivation is a possible mechanism for the drug interaction between felbamate and carbamazepine. J Pharmacol Exp Ther2003;305:1251-1262.
43.
Egnell AC, Eriksson C, Albertson N, Houston B, Boyer S: Generation and evaluation of a CYP2C9 heteroactivation pharmacophore. J Pharmacol Exp Ther2003;307:878-887.
44.
Kenworthy KE, Bloomer JC, Clarke SE, Houston JB: CYP3A4 drug interactions: correlation of 10 in vitro probe substrates. Br J Clin Pharmacol1999;48:716-727.
45.
Mayhew BS, Jones DR, Hall SD: An in vitro model for predicting in vivo inhibition of cytochrome P450 3A4 by metabolic intermediate complex formation. Drug Metab Dispos2000;28:1031-1037.
