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Assay Development

The common assay formats can include biochemical and cell-based assays. Biochemical assays to known targets are preferred due to their higher specificity, while cell-based assays are useful for phenotypes in which no specific protein target has yet been identified.


Biochemical Assays

These assays employ an enriched protein target in a defined biochemical reaction in vitro.

Receptor-ligand binding: This assay is used to select bioactive compounds that control recognition, activation, and cellular responses to ligand binding to specific receptors.  Receptors currently represent more than 60% of existing drug targets. Commonly used assays for receptors include fluorescence polarization (FP) and homogenous time-resolved fluorescence (HTRF) competition assays. Functional assays for G-protein coupled receptor (GPCR) and ion channels can also be performed.

Enzyme assays: This type of assay is used to identify bioactive compounds that inhibit a specific enzyme-mediated biological reaction.  The reaction usually is assayed by detecting chemical amounts of products produced with time.  In some cases, coupled enzymes can be used to detect the reaction if the original substrates or products are not able to be readily detected.

Protein-protein interactions: This assay is used to discovery the inhibitors that can interfere with a specific protein-protein interaction. Commonly used methods in the laboratory include homogenous time-resolve fluorescence (HTRF) competition assays and enzyme-linked immunosorbent assay (ELISA).  Protein-protein interactions are often difficult to disrupt with the compounds in existing small molecule libraries and are therefore not usually the first choice for drug research.

Cell-based Assays

These assays employ whole living cells and measure specific phenotypic responses.  Because whole cells are used, the particular pathways involved in the phenotypic response may not be as well defined, and thus can lead to artifacts in screening. 

Growth inhibition: This cell-based assay measures the compound interference with cell viability and proliferation. Typically, cells are seeded into microtiter plates (96- or 384-well) at subconfluent densities and incubated with compounds from the library.  After a further incubation period, the cell viability and density is assessed quantitatively using fluorimetric methods such as AlamarBlue dye (BioSource), or CellTiter kits (Promega).

Reporter gene: This assay is used to detect compounds that affect transcriptional control of specific genes or pathways.  Transcriptional activity is assessed by creating stable cell lines expressing a reporter gene under the control of the promoter of interest.   Commonly used reporters are b-galactosidase (LacZ), luciferase (Luc), b-glucuronidase (GUS), b-lactamase, and alkaline phosphatase (AP).

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