Multiplexing a High-Throughput Liability Assay to Leverage Efficiencies

Abstract

In order to identify potential cytochrome P-450 3A4 (drug-metabolizing enzyme) inducers at an early stage of the drug discovery process, a cell-based transactivation high-throughput luciferase reporter assay for the human pregnane X receptor (PXR) in HepG2 cells has been implemented and multiplexed with a viability end point for data interpretation, as part of a Lead Profiling portfolio of assays. As a routine part of Lead Profiling operations, assays are periodically evaluated for utility as well as for potential improvements in technology or process. We used a recent evaluation of our PXR-transactivation assay as a model for the application of Lean Thinking-based process analysis to lab-bench assay optimization and automation. This resulted in the development of a 384-well multiplexed homogeneous assay simultaneously detecting PXR transactivation and HepG2 cell cytotoxicity. In order to multiplex fluorescent and luminescent read-outs, modifications to each assay were necessary, which included optimization of multiple assay parameters such as cell density, plate type, and reagent concentrations. Subsequently, a set of compounds including known cytotoxic compounds and PXR inducers were used to validate the multiplexed assay. Results from the multiplexed assay correlate well with those from the singleplexed assay formats measuring PXR transactivation and viability separately. Implementation of the multiplexed assay for routine compound profiling provides improved data quality, sample conservation, cost savings, and resource efficiencies.

Multiplexing fluorogenic esterase-based viability assay with luciferase assays

“…Several protocols, which combine resazurin-based cell viability assay or protease-based viability assay with luciferase assay, have been developed to enable multiplex determination of cell viability (number) and luciferase activity [[6], [7], [8]]. Although these protocols work well for assays monitoring relatively slow processes, such as changes of mRNA stability or transcriptional activity, they are not suitable for monitoring faster processes, including protein degradation, due to the relatively slow conversion of resazurin to fluorescent resorufin (usually >1 h) or Gly-Phe-AFC (7-amino-4-trifluoromethylcoumarin) protease substrate (>30 min) within live cells.…”

High-Throughput Screening Reveals Alsterpaullone, 2-Cyanoethyl as a Potent p27Kip1 Transcriptional Inhibitor

“…The final cycloheximide concentration was 200 μM and the final DMSO concentration was 0.12%. The assay plates were then incubated overnight at 37°C, 5% CO 2 , 95% relative humidity followed by alamar blue cytotoxicity assay (Life Tech, #DAL1100) and luminescence luciferase reporter activity assay (Envision HTS microplate reader, PerkinElmer, Model 2102) with SteadyLite HTS reagent (PerkinElmer, #6016981) [30], [31]. Briefly, after overnight incubation, each well of the assay plates received diluted alamar blue reagent (1-to-12 dilution with DPBS (Life Tech, #14190) at 5 μl/well and incubated for an additional hour at 37°C, followed by 5 minute room temperature incubation to cool down.…”

Tools and Strategies for the Assessment of Enzyme Induction in Drug Discovery and Development

“…In general, because of differences in transfection effi ciency between assays, the transient method is subject to more assay -to -assay variability. Some groups have reported the use of batch transfection with subsequent cryopreservation as a method to reduce variability Herbst et al, 2009 ). The production of stable cell lines is limited by the number of antibiotic selection markers that can be incorporated within a single cell line, often just two.…”

“…The enzymatic reaction catalyzed by luciferase is quite effi cient, thus providing a very sensitive method for quantifi cation. This sensitivity has allowed the development of cell -based reporter gene assays based on luciferase in a 384 -well format (Herbst et al, 2009 ). As the luciferase protein is commonly expressed in the intracellular compartment, assays utilizing luciferase require a cell lysis step before analysis.…”