Multiplexing a high-throughput liability assay to leverage efficiencies.
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.
Herbst
John
J
Lead Discovery, Profiling & Compound Management, Research and Development, Bristol-Myers Squibb, 5 Research Parkway, Wallingford, CT 06492, USA.
Anthony
Monique
M
Stewart
Jeremy
J
Connors
David
D
Chen
Taosheng
T
Banks
Martyn
M
Petrillo
Edward W
EW
Agler
Michele
M
D045744:Cell Line, Tumor; D015925:Cryopreservation; D051544:Cytochrome P-450 CYP3A / Q000096:biosynthesis*; D004305:Dose-Response Relationship, Drug; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D004526:Efficiency; D004790:Enzyme Induction / Q000187:drug effects; D006801:Humans; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D000077297:Pregnane X Receptor; D011987:Receptors, Steroid / Q000096:biosynthesis / Q000235:genetics; D012015:Reference Standards; D015203:Reproducibility of Results; D013329:Structure-Activity Relationship; D014162:Transfection
D000077297:Pregnane X Receptor; D011987:Receptors, Steroid; D008156:Luciferases; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human