T. Xu, A. Kirkpatrick, S. Ripp, D. Close
Keywords: luciferase, bioluminescence, fluorescence, drug discovery, high-throughput screening, adme/Tox
Summary:Over 98% of novel compounds selected for further study in the discovery and pre-clinical screening phases of the pharmaceutical development pipeline will eventually fail. This results in the unnecessary expenditure of hundreds of millions of dollars and untold hours of personnel time that must be recouped from the few drugs that are successfully brought to market. One of the underlying problems driving this inefficiency is the prevalent use of endpoint assays at these stages. While real-time testing formats exist, their high costs prohibit their use under the high-throughput conditions required in the early stages of the development pipeline. Therefore, while using lower-cost endpoint assays is economically necessary, it remains problematic for discovery-based applications where the timing and duration of treatment effects are not known a priori. Historically, fluorescent reporter systems have been used to overcome the destructive nature of bioluminescent or antibody-based screening approaches as a means for addressing this issue, despite this approach resulting in decreased informational capacity due to autofluorescence and the persistent nature of the fluorescent protein following expression. To overcome these limitations, we developed a synthetic luciferase system that genetically encodes both its luciferase and luciferin components to enable ‘autobioluminescent’ expression without the need for external stimulation. Cells endowed with the autobioluminescent phenotype could be programmed to continuously modulate their output signal in response to real-time metabolic activity dynamics or to autonomously activate/deactivate bioluminescence in response to biomarker expression. The system successfully demonstrated the self-reporting of endocrine disrupting activity against a library of 76 agonistic and antagonistic estrogenic endocrine disruptor chemicals with reproducible half maximal effective concentration (EC50) values. For model compounds, such as 17B-estradiol, the system demonstrated an EC50 value (7.9 × 10-12 M) comparable to that of the current EPA-approved HeLa-9903 firefly luciferase-based estrogen receptor transcription assay (4.6 × 10-12 M). Similarly, when engineered to selectively report the activation of specific detoxification pathways the system self-identified the timing, magnitude, and duration of biomarker expression in response to compound treatment over a 24-hour period without human intervention. In toxicological testing experiments the effect of increasing compound treatment doses could be observed continuously for 48-hours to identify which treatment levels were toxic, which imparted transient effects that were overcome following compound metabolism, and the timing and duration of these events. By eliminating the need for sample destruction or external stimulation, 5 samples could be used to replace the equivalent of 12,000 bioluminescent endpoint assays. This increased throughput 307x per plate when testing was performed in the 1,536-well format. This synthetic luciferase-based monitoring approach provides the increased informational capacity afforded by bioluminescent reporter systems while maintaining the non-destructive, lower operational cost, and amenability to automated high-throughput applications of fluorescent systems. Using this approach to enable lower-cost, higher-throughput, real-time monitoring at the discovery and pre-clinical stages of the drug development pipeline will provide more informative data to support compound triaging and therefore help to streamline development efforts.