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Assay Development and Screening

The knowledge gained as a result of the target validation process should evolve into a schematic for the development of an assay model to be used in high throughput screening (HTS). The HTS assay plays a very important role as the first step in the process of filtering out potential ‘hits’ to be optimized as downstream drug candidates.  These assays need to fulfill several important technical criteria and at the same time be fast and efficient. Discriminating compounds based on their ability to provide some level of pharmacological intervention requires assays that are physiologically relevant, as well as robust and sensitive.

Microcalorimetry provides unique benefits as a valuable tool in the development and validation of screening assays.  Because microcalorimetry is a direct readout, label-free detection technology and does not require immobilization, it is uniquely suited to test and validate assay development strategies.

Most in-vitro screening assays utilize a surrogate readout scheme that relies on labeled molecules or some type of coupled reaction as a readout system. The biological relevance of these assay systems to the original endogenous mechanisms can be tested with Isothermal Titration Calorimetry (ITC) to compare the results and give confidence that they mimic each other appropriately. This includes all forms of ligand and receptor complexes, as well as enzyme-substrate reactions and kinetics.  Both ITC and Differential Scanning Calorimetry (DSC) can be used to evaluate the quality of expressed proteins for the appropriate post-translational properties and percent activity, as well as assess the impact of assay co-factors.

After the HTS assays have been developed, quality control of assay reagents can be preformed by DSC to ensure the integrity of the protein target reagents. This can save valuable time and expense by eliminating the possibility of assay failures during an HTS campaign.