Assay, cell-based

Active in secondary assay/cell-based assay... 

As for any new lot of material, assessments of biological activity should be performed to demonstrate the efficacy of the product. This may be achieved though the use of animal-based biological assays, cell-based assays that evaluate the material at the cellular level, and biochemical activity assays such as enzymatic action. Standards should be properly defined. Potency and efficacy for individual lots of material can therefore be defined. 

In contrast to enzyme assays, cell-based assays present the target in a more physiological milieu. With enzyme assays, it may be difficult to purify and express active kinases and phosphatases in their full-length forms and they may require the use of fusion proteins with kinase activity domains. Cell-based technologies, on the other hand, present the opportunity to express the targets with regulatory domains included. Furthermore, cell-based assays usually detect only cell-permeable inhibitors and have the potential to identify more unusual mechanisms, as described earlier. 

In vitro and in vivo biological data (e.g., enzyme or receptor assays, cell-based functional assays, efficacy animal models). 

The design of assay systems is another particularly important factor for testing the sample compounds. Assays have to be specific and sensitive. The assays used for HTS come in many forms. There are binding assays, or enzyme-based or cell-based assays. Cell-based assays have become an important test compared with other in vitro assays, as they can provide information about bioavailability, cytotoxicity and effects on biochemical pathway. Invariably, the enzyme-based and cell-based assay systems consist of receptors or mimetics of receptors (components that mimic active parts of receptors). Normally the assays are linked to an indicator that shows the ligand-receptor interaction as some forms of signal. Radioligand binding... 

Figure 6.10b shows a pattern of antagonism often observed in isolated tissue studies but not so often in cell-based assays. Saturation of uptake systems for the agonist or saturation of an adsorption site for the agonist can account for this effect. The linear portion of the regression can be used to estimate the pKB or the pA2. If there is a loss of concentration dependence of antagonism, as seen in... 

Kunkel, E. J., Plavec, L, Nguyen, D., Melrose, J., Rosier, E. S., Kao, L. T., Wang, Y., Hytopoulos, E., Bishop, A. C., Bateman, R., et al. (2004). Rapid structure-activity and selectivity analysis of kinase inhibitors by Bio Map analysis in complex human primary cell-based models. ASSAY Drug Dev. Technol. 2 431-441. 

Not all cell-based functional assays are directed against a specific molecular target. Indeed many drugs... 

Cell-Based Assays to Predict Toxicity and Resistance Aspects... 

Before an antiviral agent becomes a drug, advanced toxicity testing, pharmacological combination, and drug-interaction studies are needed. The use of new cell-based assays that can predict mitochondrial toxicity, lactic acidosis, peripheral neuropathy, anemia, hypersensitivity, lipodystrophy, and other potential side effects can alleviate these issues (Stuyver et al. 2002). 

Kinases are enzymes that place a phosphate group on a serine/threonine or a tyrosine residue of a protein or peptide. All kinase reactions use ATP as the phosphate source. Therefore there have been assays developed that monitor the loss or gain of the peptide/protein substrate (LANCE, ULight) [23], the loss of ATP (easylite luminescence kinaseGlo, Perkin Elmer) [20], or the gain of ADP (Tran-screener TR-FRET) [24]. Many of these formats are applicable to cell based assays. 

Cell based assays for NRs range from reporter gene assays to in vivo recruitment assays. The most reported of these is the GAL4 reporter assay. This assay takes advantage of the fact that the GAL4 response element of yeast does not exist in mammalian systems. 

---