Cell-based assay optimization

The ultimate goal of lead optimization is to produce compounds that will elicit the desired cellular and organismal phenotype when dosed at appropriate concentrations. During the course of lead optimization activities it is common for pharmacologists to evaluate compounds not only using in vitro enzyme activity assays but also in cell-based assays as well. A question that often arises at this stage of drug discov-... 

Two main factors have guided the need for optimization of the early screening techniques on one hand the use of simple, quick and high-capacity cell monolayer methods, e.g., Caco-2 cell and MDCK and on the other hand the increased synthesis of more lipophilic, insoluble compounds from combinatorial libraries. This has created a vast number of different variants of cell-based assays and has resulted in variability among the data obtained. A need for optimization of as many as possible of the different parameters in order to increase the predictivity and throughput of the model has been suggested in the literature [98-100]. 

Lautraite S, Bigot-Lasserre D, Bars R et al. (2003) Optimization of cell-based assays for medium throughput screening of oxidative stress. Toxicol In Vitro. 17 207-220. 

Growth conditions in deep-well microtiter plates were optimized with respect to optimal expression of active enzymes (Fig. 2.2.1.1). The best results were obtained with an expression time of 20 h at 37 °C (Fig. 2.2.1.1, lanes 7-9). Subsequently, E. coli cells were enzymatically disrupted by lysozyme treatment, and the carboligase activity was monitored by a modified tetrazolium salt color assay [16], This color assay is based on the reduction of the 2,3,5-triphenyltetrazolium chloride (TTC) 13 to the corresponding formazan 15, which has an intense red color (Fig. 2.2.1.2A). Before screening ofa BFD variant library, substrates and products were tested in the color assay. Neither substrate, benzaldehyde 4 nor dimethoxy-acetaldehyde 8, reduced TTC 13 however, the product 2-hydroxy-3,3-dimethoxy-propiophenone 10 already caused color formation at low concentrations of 2.5-10 mM (Fig. 2.2.1.2B). Benzoin 12 as the product also gave a color change at a similar concentration (data not shown). 

Analogous to cell viability measurements, many of the same basic concepts apply to the development of cell-based assays for apoptosis. The length of incubation of cells with the test compound is among the most important issues to address and optimize. The length of incubation is important because the markers of apoptosis may be present for relatively brief transient periods and subsequently disappear as the population of cells undergoes secondary necrosis. The induction of measurable caspase activity can occur in only a few minutes or can take days, depending on the model cell line, type of inducer, and effective concentration inside the cells. 

Profile Cellomics Inc., formerly BioDx Inc., is a privately held corporation founded in October 1996. Cellomics mission is to improve the efficiency of the drug discovery process by delivering a cell-based screening platform that automates target validation and lead optimization using fluorescence-based assays. 

---