Functional cell-based assay

Directed evolution relies on the analysis of large numbers of clones to enable the discovery of rare variants with unproved function. In order to analyze these large libraries, methods of screening or selection have been developed, many of which use specialized equipment or automation. These range from the use of multichannel pipettes, all the way up to robotics, depending on the level of investment [59]. Specialized robotic systems are available to perform tasks such as colony picking, cell culture, protein purification, and cell-based assays. 

Functional in vitro assays can be whole-cell assays or animal tissue assays. For in vitro cell-based assays, recombinant molecular targets can be expressed in cell lines and linked to second-messenger systems to measure functional responses. Examples of functional... 

Secondary assays depend on the project. Where the primary screen was a cell-based assay, the secondary assay may be a radioligand competition binding assay. In other cases, such as where the primary screen was a biochemical assay, the secondary assay may be a cellular assay, and may be functional or mechanistic. One of the issues that may arise at this stage is that compounds with reasonable activity in the primary assay may not show activity in the secondary assay. There can be a number of reasons for this, including insufficient potency, inability of the compound to get into cells, or a higher intracellular concentration of the natural ligand (e.g., ATP) if the inhibitor is a competitive inhibitor. It is often necessary at this stage to prepare additional compounds in the series to get compounds of sufficient potency and/or permeability so that cellular activity can be demonstrated. 

The first functional cell-based assay for the determination of pan-histone deacetylase activity was achieved by detecting hyperacetylated histones by Western blotting. The histone acetylation level in cells offers a measure of candidate HDACI activity and has been linked to antiproliferative and cytotoxic effects. Beckers et al. used a cellular histone hyperacetylation assay for quantification of the cellular efficacy of HDACIs using the Cellomics Array Scan II platform in 96 wells [14]. The program... 

Among the biological assays, preference is given to assays that measure a direct interaction between a compound and a target, making data interpretation simpler. Therefore, radioligand binding assays and isolated enzyme assays are preferred to functional or cell-based assays. 

The final example described in this chapter is that done by Schriebej and co-workers. They used a scaffold with multivariant sites derived from shikimic acid. However, the end products of their synthesis were compact, highly functionalized structures reminiscent of alkaloids.34 An objective of this study was to produce a very large number of compounds for miniaturized cell-based assays. In fact, about 2 million compounds were made. 

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