A Genetically‐Encoded Fluorescent Assay to Detect Drug Toxicity and Genetic Stress in Living Cells

Abstract

We have created a genetically‐encoded fluorescent biosensor to detect chemically and genetically induced stress in living cells. Identifying stressed cells long before they enter the apoptotic pathway opens a therapeutic window between cell stress and death. Further, the biosensor can also be used to identify toxic side effects of drugs or lead compounds.The sensor co‐opts one pathway of the unfolded protein response as the driving mechanism to detect cell stress. Non‐canonical splicing of an intron derived from the XBP1 protein shifts into frame a bright green fluorescent protein, which indicates the onset of cell stress. Importantly, this intron is only spliced upon activation of endoplasmic reticulum stress or the unfolded protein response. Ratioing this signal with a constitutively expressed red fluorescent protein signal allows real‐time monitoring of cells stress and protein production. We have also engineered this biosensor to be reversible, allowing detection of the onset of stress as well as the amelioration of stress. This positions the biosensor to be used for testing compounds that may relieve cell stress.We applied this assay to examine the toxic side effects of cardiac glycosides and doxorubicin on cardiomyocytes derived from human induced pluripotent stem cells. Drug toxicity was detected within 24 hours of treatment in a dose dependent manner. The cardiac glycosides induced cell stress mediated through either the unfolded protein response or endoplasmic reticulum stress while doxorubicin, which inhibits the XBP1 pathway, mainly displayed cytotoxic effects. We next used this assay to detect cellular stress induced by genetic mutations. Many neurological diseases, such as Retinitis Pigmentosa, Parkinson's and Amyotrophic lateral Sclerosis (ALS), are associated with protein folding or aggregation defects which induce high levels of cellular stress. This live‐cell stress assay is capable of monitoring cellular stress induced by mutations causing Retinitis Pigmentosa (Rhodopsin P23H mutation) and ALS (SOD G86R mutation) in less than 24 hours after mutant gene expression.Lastly, multiplexing this cell stress assay with other live‐cell assays detecting second messenger signaling revealed cells experiencing either chemically or genetically induced stress had altered basal levels of cytoplasmic Ca2+ and cAMP as well as a blunted Ca2+ signaling response. Together these studies demonstrate the broad utility of this newly developed live‐cell assay for cell stress and toxicity for use in automated screening of drugs and compounds, determination of adverse drug toxicity, and analysis of disease mechanisms.Support or Funding InformationSupported by ASEE/NSF Small Business Postdoctoral Research Diversity Fellowship Program NSF Award IIP‐1552305This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.