Using ß-Lactamase and NanoLuc Luciferase Reporter Gene Assays to Identify Inhibitors of the HIF-1 Signaling Pathway.

The hypoxia-inducible factor 1 (HIF-1) is a transcriptional factor involved in the regulation of oxygen within cellular environments. In hypoxic tissues or those with inadequate oxygen concentrations, activation of the HIF-1 transcription factor allows for subsequent activation of target gene expression implicated in cell survival. As a result, cells proliferate through formation of new blood vessels and expansion of vascular systems, providing necessary nourishment needed of cells. HIF-1 is also involved in the complex pathophysiology associated with cancer cells. Solid tumors are able to thrive in hypoxic environments by overactivating these target genes in order to grow and metastasize. Therefore, it is of high importance to identify modulators of the HIF-1 signaling pathway for possible development of anticancer drugs and to better understand how environmental chemicals cause cancer. Using a quantitative high-throughput screening (qHTS) approach, we are able to screen large chemical libraries to profile potential small molecule modulators of the HIF-1 signaling pathway in a 1536-well format. This chapter describes two orthogonal cell based assays; one utilizing a ß-lactamase reporter gene incorporated into human ME-180 cervical cancer cells, and the other using a NanoLuc luciferase reporter system in human HCT116 colon cancer cells. Cell viability assays for each cell line are also conducted respectively. The data from this screening platform can be used as a gateway to study mode of action (MOA) of selected compounds and drug classes.

Transactivation and Coactivator Recruitment Assays for Measuring Farnesoid X Receptor Activity.

The farnesoid X receptor (FXR) is a nuclear receptor responsible for homeostasis of bile acids, lipids, and glucose. Compounds that alter endogenous FXR signaling can be used as therapeutic candidates or identified as potentially hazardous compounds depending on exposure doses and health states. Therefore, there is an increasing need for high-throughput screening assays of FXR activity to profile large numbers of environmental chemicals and drugs. This chapter describes a workflow of FXR modulator identification and characterization. To identify compounds that modulate FXR transactivation at the cellular level, we first screen compounds from the Tox21 10 K compound library in an FXR-driven beta-lactamase reporter gene assay multiplexed with a cell viability assay in the same well of the 1536-well plates. The selected compounds are then tested biochemically for their ability to modulate FXR-coactivator binding interactions using a time-resolved fluorescence resonance energy transfer (TR-FRET) coactivator assay. The assay results from the workflow can be used to prioritize compounds for more extensive investigations.