Target-based biochemical assays

The major advantage of the antisense RNA modulated cell-based assays is that the target specifically sensitized cells would respond to lower concentrations of target-specific inhibitors in libraries of pure compounds or natural product extracts that could not be as easily identified if a traditional cell-based growth inhibition screen were performed. In contrast to target-based biochemical assays (as described in Section 7.2.1), the hit compounds discovered in antisense... 

The approaches used to discover antibacterial drugs include cell-free target-based biochemical screens (Section 7.2.1), cell-based assays combined with underexpression (cell sensitization, Section 7.2.2) or overexpression (cell resistance, Section 7.2.3), and other cell-based assays followed by sophisticated molecular biology and genetic analyses (Section 7.2.4). These approaches are discussed in detail in the following sections. 

For testing the biological activity of such a large number of molecules, development of fast assays and with a read-out as simple as possible is essential. These assays can be performed on molecular targets (biochemical assays) or target cell (cell-based assays). 

To study drug-receptor/enzyme interaction, it is not always convenient or appropriate to use a living system of the target receptor. Instead, biochemical assays can be devised to mimic the target. Very often, the assays use multicolor luminescence or fluorescence-based reagents. In this way, the reaction path can be followed in space and time to enable quantitative evaluation of the reaction. 

ALIS reports only compounds that bind directly to the target of interest, preventing false positives that arise from off-target activity or interactions with substrates or other reagents. Since ALIS directly identifies bound components by MS, the incidence of false positives based on bulk effects and non-specific binding is lower than that of biochemical assays that yield a secondary readout of activity. 

With more than 500 members, the proteases constitute one of the largest families of enzymes in the human genome and are an important class of targets for drug discovery. Biochemical assays based on fluorescence readouts are the most frequently used technologies to elaborate the enzymatic mechanisms of proteases and to test the inhibitory potencies of drug-like chemical compounds. These assay formats exist for all classes of proteases and substrate specificities. 

The process of identifying new chemical entities (NCEs) often involves the use of cell-based or biochemical assays by which the effects of new compounds on a specific target of interest are monitored. Transformed cell lines have been the traditional mainstays of assay development since the beginning of high-throughput screening (HTS) in the early 1980s. 

Many of the natural compounds isolated and characterised in the past underwent a very limited biological profiling, i.e. they were usually tested only in antibacterial and/or antifungal assays. This applies particularly to the thousands of natural products isolated and characterised by academic groups. Testing of such compounds in biochemical target-based assays or in modern phenotypical cell-based assays should reveal new biological activities and many new applications. 

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