Hypoxia-inducible factor-1 (HIF-1) is the major hypoxia-regulated transcription factor that regulates cellular responses to low oxygen environments. HIF-1 is composed of two subunits: hypoxia-inducible HIF-1α and constitutively-expressed HIF-1β. During hypoxic conditions, HIF-1α heterodimerizes with HIF-1β and translocates to the nucleus where the HIF-1 complex binds to the hypoxia-response element (HRE) and activates expression of target genes implicated in cell growth and survival. HIF-1α protein expression is elevated in many solid tumors, including those of the cervix and brain, where cells that are the greatest distance from blood vessels, and therefore the most hypoxic, express the highest levels of HIF-1α. Therapeutic blockade of the HIF-1 signaling pathway in cancer cells therefore provides an attractive strategy for development of anticancer drugs. To identify small molecule inhibitors of the HIF-1 pathway, we have developed a cell-based reporter gene assay and screened a large compound library by using a quantitative high-throughput screening (qHTS) approach.

The endoplasmic reticulum (ER), a multifunctional organelle, plays a central role in cellular signaling, development, and stress response. Dysregulation of ER homeostasis has been associated with human diseases, such as cancer, inflammation, and diabetes. A broad spectrum of stressful stimuli including hypoxia as well as a variety of pharmacological agents can lead to the ER stress response. In this study, we have developed a stable ER stress reporter cell line that stably expresses a β-lactamase reporter gene under the control of the ER stress response element (ESRE) present in the glucose-regulated protein, 78 kDa (GRP78) gene promoter. This assay has been optimized and miniaturized into a 1536-well plate format. In order to identify clinically used drugs that induce ER stress response, we screened approximately 2800 drugs from the NIH Chemical Genomics Center Pharmaceutical Collection (NPC library) using a quantitative high-throughput screening (qHTS) platform. From this study, we have identified several known ER stress inducers, such as 17-AAG (via HSP90 inhibition), as well as several novel ER stress inducers such as AMI-193 and spiperone. The confirmed drugs were further studied for their effects on the phosphorylation of eukaryotic initiation factor 2α (eIF2α), the X-box-binding protein (XBP1) splicing, and GRP78 gene expression. These results suggest that the ER stress inducers identified from the NPC library using the qHTS approach could shed new lights on the potential therapeutic targets of these drugs.

The nuclear receptor, estrogen-related receptor alpha (ERRα; NR3B1), plays a pivotal role in energy homeostasis. Its expression fluctuates with the demands of energy production in various tissues. When paired with the peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α), the PGC/ERR pathway regulates a host of genes that participate in metabolic signaling networks and in mitochondrial oxidative respiration. Unregulated overexpression of ERRα is found in many cancer cells, implicating a role in cancer progression and other metabolism-related diseases. Using high throughput screening assays, we screened the Tox21 10K compound library in stably transfected HEK293 cells containing either the ERRα-reporter or the reporter plus PGC-1α expression plasmid. We identified two groups of antagonists that were potent inhibitors of ERRα activity and/or the PGC/ERR pathway: nine antineoplastic agents and thirteen pesticides. Results were confirmed using gene expression studies. These findings suggest a novel mechanism of action on bioenergetics for five of the nine antineoplastic drugs. Nine of the thirteen pesticides, which have not been investigated previously for ERRα disrupting activity, were classified as such. In conclusion, we demonstrated that high-throughput screening assays can be used to reveal new biological properties of therapeutic and environmental chemicals, broadening our understanding of their modes of action.