High-throughput and combinatorial gene expression on a chip for metabolism-induced toxicology screening.

Differential expression of various drug-metabolizing enzymes (DMEs) in the human liver may cause deviations of pharmacokinetic profiles, resulting in interindividual variability of drug toxicity and/or efficacy. Here, we present the 'Transfected Enzyme and Metabolism Chip' (TeamChip), which predicts potential metabolism-induced drug or drug-candidate toxicity. The TeamChip is prepared by delivering genes into miniaturized three-dimensional cellular microarrays on a micropillar chip using recombinant adenoviruses in a complementary microwell chip. The device enables users to manipulate the expression of individual and multiple human metabolizing-enzyme genes (such as CYP3A4, CYP2D6, CYP2C9, CYP1A2, CYP2E1 and UGT1A4) in THLE-2 cell microarrays. To identify specific enzymes involved in drug detoxification, we created 84 combinations of metabolic-gene expressions in a combinatorial fashion on a single microarray. Thus, the TeamChip platform can provide critical information necessary for evaluating metabolism-induced toxicity in a high-throughput manner.

Rapid and quantitative measurement of metabolic stability without chromatography or mass spectrometry.

Metabolic stability measurements are a critical component of preclinical drug development. Available measurement strategies rely on chromatography and mass spectrometry, which are expensive and labor intensive. We have developed a general method to determine the metabolic stability of virtually any compound by quantifying cofactors in the mechanism of cytochrome P450 enzymes using fluorescence intensity measurements. While many previous studies have shown that simple measurements of cofactor depletion do not correlate with substrate conversion (i.e., metabolic stability) in P450 systems, the present work employs a reaction engineering approach to simplify the overall rate equation, thus allowing the accurate and quantitative determination of substrate depletion from simultaneous measurements of NADPH and oxygen depletion. This method combines the accuracy and generality of chromatography with the ease, throughput, and real-time capabilities of fluorescence.

P450cam biocatalysis in surfactant-stabilized two-phase emulsions.

Cytochrome P450 monooxygenases (P450s) are powerful biocatalysts that have the ability to oxidize a broad range of substrates, often at non-reactive carbon centers. However, incorporation of P450s into synthetic schemes has so far been limited to a few whole-cell transformations. P450 substrates are often hydrophobic and have low water solubility, limiting the amount of product that can be produced. To help overcome this limitation, we have examined P450cam activity in two-phase hexane/water emulsions with and without the anionic surfactant, bis(2-ethylhexyl) sulfosuccinate sodium salt (AOT). Hydroxylation of camphor to hydroxycamphor by the three- component P450cam system was chosen as the model reaction, and regeneration of NADH was accomplished with yeast alcohol dehydrogenase (YADH). P450cam was activated in the surfactant-free emulsions, and addition of AOT improved the activity even further, at least over the range of camphor concentrations for which initial rates were readily measurable in all media. The largest observed rate enhancement was 4.5-fold. Nearly 50-times more product was formed in the surfactant-stabilized emulsions than was achieved in aqueous buffer, with total turnover numbers reaching 28,900 for P450cam and 11,800 for YADH. In the absence of surfactant, the two-phase reaction appeared to be mass-transfer limited, while inclusion of AOT alleviated transport limitations and/or afforded a larger interfacial area for P450 activation. The oxidation of hydroxycamphor to 2,5-diketocamphane was also observed, owing to the large concentration of hydroxycamphor relative to camphor in the aqueous phase of the two-phase emulsion. This competing reaction was accompanied by the uncoupled oxidation of NADH (i.e., NADH oxidation without formation of 2,5-diketocamphane), which reduced the availability of NADH for camphor oxidation and further limited the yield of hydroxycamphor in the two-phase emulsions. These results indicate that a surfactant-stabilized two-phase emulsion is a promising reaction medium for practical P450 biocatalysis, although its effectiveness for a given P450/substrate combination can depend on several factors, including competitive or sequential reactions, product inhibition, and NAD(P)H uncoupling.

Cell-based screen of HMG-CoA reductase inhibitors and expression regulators using LC-MS.

We present an integrated consolidation of previously reported methods for screening hydroxymethylglutaryl-coenzyme A reductase (HMGR) inhibitors in 96-well microtiter plates with rapid workup using established mammalian cell lines and liquid chromatography-mass spectrometry analysis. Inhibitors as well as expression regulators of HMGR (inducers or repressors) can be screened. To validate the method, three competitive inhibitors of HMGR (lovastatin, simvastatin, and atorvastatin), as well as a potent sterol repressor of HMGR synthesis (25-hydroxycholesterol), were assayed on two cell lines: HepG2, a human hepatic derived cell line, and L cells, a subline of NCTC clone 929 mouse fibroblasts. The direct inhibition of HMGR by statins, induction of HMGR synthesis by the same statins following incubation with the cells, and repression of HMGR synthesis by 25-hydroxycholesterol were confirmed.