Assay “readout” technologies fluorescence

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 truth most likely lies somewhere in between. Bender [67] published the most quantitative study to date on the success of HTS at Novartis. Several conclusions could be drawn. Particular target types and assay technologies have a great impact on screening success, and this was not always correlated to the number of identifying hits in the HTS runs. For assay formats used a minimum of five times, LC/MS readouts succeed 83% of the time, followed by FP assays, which succeed in 72% of the cases. TR-FRET showed a success rate of 70%, with FLIPR assays (61%), fluorescence intensity readouts (59%), and AlphaScreen (60%) performing... 

Fluorescent-based flux or membrane potential assays provide superior throughput compared to traditional electrophysiology-based screening technologies. For fluorescent-based membrane potential assays, however, the response to increasing channel activity can be non-linear due to the inherent non-linear relationship between channel activity and membrane potential (Fig. 4A). This prevents an accurate readout of efficacy and reduces the reso-... 

Trends in biochemical screening assays seem to favor the use of multi-function PMT-based readers that allow for various MTP well densities (96, 384, and 1536 well plates), can handle a number of readout formats such as prompt fluorescence, luminescence, fluorescence polarization, time-gated fluorescence, and luminescent oxygen channeling or AlphaScreen. Examples of this type including the Perkin Elmer EnVision, TECAN-Ultra, BMG FluoStar, and LJL Analyst GT can be employed for a variety of the assay technologies described above. 

In addition to the general criteria discussed above, each assay technology can also suffer from technology-specific interference of chemical compounds. Auto-fluorescent compounds can interfere with any fluorescence-based readout. In assays that use the biotin-streptavidin interaction, biotin analogs are potential false positives. 

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