High throughput P450 inhibition screens in early drug discovery.

This review of high throughput (HT) P450 inhibition technologies and their impact on early drug discovery finds the field at a mature stage. The relationship between P450 inhibition and drug-drug interactions is well understood. A wide variety of P450 inhibition detection technologies are readily available off-the-shelf, but what seems still to be missing is a general agreement on how much weight one should give to the various types of early discovery HT P450 inhibition data. Method-dependent potency differences are a cause of concern, and to resolve this issue the authors advocate calibration of the HT methods with a large set of marketed drugs.

In silico prediction of drug safety: despite progress there is abundant room for improvement.

Predictive models for drug safety are crucial for helping to avoid costly late-stage failures. We review recent work on models for genotoxicity, liver toxicity, CYP450 inhibition and cardiotoxicity. These models have improved somewhat in recent years, and research has expanded into new frontiers, such as the prediction of liver toxicity. However, much more needs to be done.:

Detection of beta-lactamase reporter gene expression by flow cytometry.

BACKGROUND: Flow cytometry of gene expression in living cells requires accurate, sensitive, nontoxic fluorescent indicators capable of detecting transcription of specific genes. This is typically achieved by using genes that encode fluorescent proteins or enzymes coupled to promoters of interest. The most commonly used reporters are green fluorescent protein and beta-galactosidase (lacZ). In this study, we characterized the performance of a cell-permeant, ratiometric, beta-lactamase substrate, coumarin cephalosporin fluorescein (CCF2/AM). We compared its characteristics with that of the beta-galactosidase/fluorescein di-beta-D-galactopyranoside reporter system. METHODS: Jurkat cell lines were generated for beta-lactamase and beta-galactosidase reporters with the use of similar plasmid constructs. Rare event flow cytometric detection for the beta-galactosidase and beta-lactamase reporters were assayed by using mixed populations of negative (WT) and positive (constitutively expressing) cells for each reporter. To determine sensitivity at low reporter copy number, we measured the activity of an unstimulated inducible promoter and detected positive events as a function of substrate incubation time. Technical issues related to data processing and optical configuration are also presented. RESULTS: The low population coefficients of variation afforded by ratiometric detection of the beta-lactamase system improved the statistical performance of the assay in comparison with a single-dye, intensity-based assay, leading to markedly improved detection for low copy number and rare events. At low levels of gene expression, beta-lactamase was detected with approximately 10-fold higher confidence than was beta-galactosidase. In rare event detection experiments, cells expressing high levels of beta-lactamase proteins were reliably detected at frequencies of 1:10(6) compared with about 1:10(4) for beta-galactosidase. CONCLUSION: The ratiometric fluorescence readout of the beta-lactamase system based on fluorescence resonance energy transfer allowed more sensitive and accurate detection of gene expression than the currently available beta-galactosidase substrates. Further, the cell-permeant nature of the substrate improved experimental convenience. These properties facilitated cell engineering and enabled a variety of applications including selection of rare cells from large populations and measurement of low-expressing or downregulated genes.