Probing Chemical Processes in Living Cells: Applications for Assay and Screening of Chemicals that Disrupt Cellular Signaling Pathways

Abstract

Abstract Methods of analysis for bioactive substances that are based on cellular signaling pathways are discussed and compared with the binding assay and bioassay. For nondestructive analysis of chemical processes in living cells, we have developed some general methods and new intracellular fluorescent probes for detecting 1) second messengers, cGMP, diacylglycerol and phosphatidylinositol-3,4,5-triphosphate, 2) protein phosphorylation, 3) protein conformational changes, and 4) protein-protein interactions in live cells under a confocal laser microscope. The approaches for the present probe developments are use of fluorescence resonance energy transfer for reporting binding of substrates (analytes) to molecular recognition domains in dual-fluorophore conjugated probe molecules, and use of protein splicing chemistry for detecting protein-protein interactions. Key molecules and steps of cellular signaling pathways were visualized in relevant live cells using developed fluorescent probe molecules. These probes were found of general importance not only for fundamental biology studies, but also for assay and screening methods for chemicals that inhibit or facilitate cellular signaling pathways. Changes in cellular signals were thereby observed in nongenomic pathways of steroid hormones upon treatment of the target cells with steroid hormones and xenoestrogens. This method of analysis appears to be a rational approach to high-throughput prescreening of biohazardous chemicals such as endocrine disrupting chemicals that disrupt these cellular signaling pathways.