Abstract
Modern drug discovery workflows require assay systems capable of replicating the complex interactions of multiple tissue types, but that can still function under high throughput conditions. In this work, we evaluate the use of substrate-free autobioluminescence in human cell lines to support the performance of these assays with reduced economical and logistical restrictions relative to substrate-requiring bioluminescent reporter systems. The use of autobioluminescence was found to support assay functionality similar to existing luciferase reporter targets. The autobioluminescent assay format was observed to correlate strongly with general metabolic activity markers such as ATP content and the presence of reactive oxygen species, but not with secondary markers such as glutathione depletion. At the transcriptional level, autobioluminescent dynamics were most closely associated with expression of the CYP1A1 phase I detoxification pathway. These results suggest constitutively autobioluminescent cells can function as general metabolic activity bioreporters, while pairing expression of the autobioluminescent phenotype to detoxification pathway specific promoters could create more specific sensor systems.
Highlights
Drug discovery relies on a combination of in vitro cell culture-based and in vivo whole animal-based models to identify, validate, and ensure the safety of promising therapeutic agents.The upstream, in vitro testing component of this system serves as a high throughput triage, with an average of 10,000 molecules screened for each new lead compound developed [1]
T47D metabolism was significantly impacted by treatment levels ≥ 1 μM, with no difference if the compound was dosed directly or pre-metabolized by the HepG2 cell line
Pre-metabolism by the T47D cell line caused HepG2 metabolism to increase relative to the effect of directly dosing the cells, while the opposite was observed for T47D cells treated with Doxorubicin hydrochloride pre-metabolized by HepG2 cells
Summary
Drug discovery relies on a combination of in vitro cell culture-based and in vivo whole animal-based models to identify, validate, and ensure the safety of promising therapeutic agents.The upstream, in vitro testing component of this system serves as a high throughput triage, with an average of 10,000 molecules screened for each new lead compound developed [1]. A significant stumbling block towards this end has been the lack of complexity and parallel systems interaction provided by these tests relative to their downstream, in vitro counterparts This presents a challenge whereby increasing the complexity of the system adds cost and decreases throughput, and decreasing the complexity lowers its predictive ability and allows false positive compounds to proceed to the more expensive in vivo testing stage. This conundrum requires that complex systems be endowed with low-cost, high throughput technologies in order to provide both the scientific and economic power required to identify promising lead compounds and quickly move
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