Cell-based reporter assays

Plants are not different from other natural product samples in that they too tend to interfere with various screening formats in nonspecific ways as nuisance compounds displaying unwanted color, inherent fluorescence, promiscuous or aggregate behaviors, detergent-like activities, or toxicity (Feng et al., 2005 Appleton, Buss, and Butler, 2007). Biochemical assays (cell-free defined systems) are notoriously sensitive to such interference by natural product extracts. Cell-based reporter assays and cell-based so-called phenotypic screens always require parental cell controls to determine extract toxicity. However, plants contain their own sets of components that are problematic to screening assays and compound identification. 

Dai, J., Liu, Y., Jia, H Zhou, Y.-D., and Nagle, D.G. (2007) Benzochromenones from the marine crinoid Comantheria rotula inhibit hypoxia-inducible factor-1 (HIF-1) in cell-based reporter assays and differentially suppress the growth of certain tumor cell lines./. Nat. Prod., 70,1462-1466. 

S. W. (2002) A cell-based reporter gene assay for determining induction of CYP3A4 in a high-volume system. Journal of Pharmacology and Experimental Therapeutics, 303, 412-423. 

Reporter gene assay (RGA), e.gv lucHerase, SEAP Secondary m essengers, e.g., Ca24(FLIPR), cAMP, IP3 J Cell-based ELISA e.g.,QytoBlDt Cell based physiological assay... 

A number of different competitive cell-free and cell-based binding assays under static and hydrodynamic flow conditions have been used to obtain affinity data for selectin ligands. In addition to the fact that different positive controls have been used, this makes a direct comparison of reported binding affinities difficult. Therefore, in this chapter, we quote relative affinities wherever possible (Section 16.4.3). Another problem that has a negative effect on assay reliabihty has been encountered in cases where acidic ion exchange resins are used in the final step of the antagonist synthesis [170]. Small amounts of polyanions released from the resin were found to be potent selectin inhibitors, especially for P-selectin. These polyanions are difficult to remove and are not detectable by routine analysis. As a result, published assay data for P-selectin antagonists should be considered with caution. 

Yeast reporter gene assays and MCF-7 cell-based proliferation assays (E-screen) are particularly popular. The E-screen, in which proliferation of human breast cancer cells (MCF-7) is measured as a response to estrogen, has also been used to determine the estrogenicity of sewage effluent and surface water (Kdrner et al., 2001). 

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