Category: Assay Development and Screening
Toxic side effects of drugs and lead compounds represents a major hurdle during the drug discovery processes. Nearly two-thirds of drugs fail prior to Phase II clinical testing, many due to adverse toxicity. Early detection of adverse toxicity of drugs or lead compounds prior to clinical trials would aid in rapid, cost-effective drug development; yet few tools exist to test for cellular toxicity in disease relevant cell types early in drug development. Those that due often only report live/dead cells, lacking valuable information such as toxicity mechanisms and dose dependencies. We have created a genetically-encoded fluorescent assay to detect chemically and genetically induced stress in living cells. We applied this assay to examine the toxic side effects of a class of chemotherapeutics known as receptor tyrosine kinase (RTK) inhibitors and doxorubicin on cell stress. A major adverse effect of both RTK inhibitors and doxorubicin is drug induced cardiotoxicity. Utilizing our cell stress assay we analyzed the effect of these chemotherapeutics on cellular stress in both HEK293 cells and cardiomyocytes derived from human induced pluripotent stem cells. Drug toxicity was detected within 24 hours of treatment in a dose dependent manner. The various RTK inhibitors displayed different dose dependent effects on cell stress and viability, suggesting possible unique stress inducing mechanisms from a single drug class. This rapid detection of drug induced toxicity can be readily adapted to automated screening in a variety of biologically relevant cell types to allow for early stage detection of adverse drug or lead compound side effects. We next used this assay to detect cellular stress induced by genetic mutations. Many neurological diseases, such as 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), Parkinson’s disease (alpha synuclein A53T mutation), and ALS (SOD G86R mutation) less than 24 hours after mutant gene expression. 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.
Jake Quinn– Commercialization Manager, Montana Molecular, BOZEMAN, MT