The Identification of Pivotal Transcriptional Factors Mediating Cell Responses to Drugs With Drug-Induced Liver Injury Liabilities

Abstract

Drug-induced liver injury (DILI) is a leading cause of drug attrition during drug development and a common reason for drug withdrawal from the market. The poor predictability of conventional animal-based approaches necessitates the development of alternative testing approaches. A body of evidence associates DILI with the induction of stress-response genes in liver cells. Here, we set out to identify signal transduction pathways predominantly involved in the regulation of gene transcription by DILI drugs. To this end, we employed ATTAGENE's cell-based multiplexed reporter assay, the FACTORIAL transcription factor (TF), that enables quantitative assessment of the activity of multiple stress-responsive TFs in a single well of cells. Homogeneous reporter system enables quantitative functional assessment of multiple transcription factors. Nat. Methods 5, 253-260). Using this assay, we assessed TF responses of the human hepatoma cell line HepG2 to a panel of 64 drug candidates, including 23 preclinical DILI and 11 clinical DILI compounds and 30 nonhepatotoxic compounds from a diverse physicochemical property space. We have identified 16 TF families that specifically responded to DILI drugs, including nuclear factor (erythroid-derived 2)-like 2 antioxidant response element, octamer, hypoxia inducible factor 1 alpha, farnesoid-X receptor, TCF/beta-catenin, aryl hydrocarbon receptor, activator protein-1, E2F, early growth response-1, metal-response transcription factor 1, sterol regulatory element-binding protein, paired box protein, peroxisome proliferator-activated receptor, liver X receptor, interferone regulating factor, and P53, and 2 promoters that responded to multiple TFs (cytomegalovirus and direct repeat 3/vitamin D receptor). Some of TFs identified here also have previously defined role in pathogenesis of liver diseases. These data demonstrate the utility of cost-effective, animal-free, TF profiling assay for detecting DILI potential of drug candidates at early stages of drug development.

Evaluating biological activity of compounds by transcription factor activity profiling

“…Twenty-four hours later, cells were rinsed and incubated for another 24 hours with the evaluated compounds in a Dulbecco’s modified Eagle’s medium (DMEM) growth medium supplemented with 1% charcoal-stripped fetal bovine serum and antibiotics. Total RNA was isolated, and the RTU activity profiles were assessed by consecutive steps of RT-PCR amplification, Hpa I digest, and CE, as described ( 5 , 6 ).…”

“…Therefore, we have developed a multiplex reporter system (the FACTORIAL) enabling a parallel assessment of the activity of multiple TFs in a single well of cells ( 4 ). We used this system to analyze TF responses to environmental chemicals and drugs ( 5 , 6 ).…”

Vascular homeostasis at high‐altitude: role of genetic variants and transcription factors

“…Of consequence, research on the role of TFs in influencing drug response is increasing. 117 , 118 For instance, one mechanism of action of aminophylline involves recruitment and activation of HDACs, 119 allele-specific HDACs binding to genetic variants may alter drug response in HAPE. Likewise, acetazolamide controls transcriptional activation of several TFs namely, AP-1, HIF, Heat shock factor (HSF), Nuclear factor-kappa B (NF-κB), Nuclear factor erythroid 2-related factor 2 (NRF2), Tumor suppresor p53 (p53), and STAT3.…”

“…Such insights from pharmacogenomics research will help to characterize genetic determinants effecting drug response to HAPE; paving way to personalized medicines. Of consequence, research on the role of TFs in influencing drug response is increasing 117 , 118 . For instance, one mechanism of action of aminophylline involves recruitment and activation of HDACs, 119 allele‐specific HDACs binding to genetic variants may alter drug response in HAPE.…”

Assessing risks and mechanisms of idiosyncratic drug toxicity by fingerprints of cell signaling responses

“…We used the FACTORIAL platform in a previous study to survey TF responses to DILI-concern drug candidates( 12 ). We found several preferentially responding to DILI-concern drugs TFs, but none of the individual TFs alone could unequivocally predict DILI liability.…”