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Abstract for Bioluminescent Approaches for In Vitro ADMET
Michael Curtin, James J. Cali, Mary Sobol, Dongping Ma, John
Shultz, Michael Valley
Promega Corporation, 2800 Woods Hollow Rd., Madison, WI 53711
ADMET targets were coupled to firefly luciferase in assays that
used the convenience of light output as a readout. In a cell-based
approach test compounds were tested for glutathione depletion, P450
transcriptional induction and induction of P450 enzyme activity.
Inductions of P450 enzyme activities with selective luminogenic
substrates were measured and observed as markers for identifying
ligands for PXR, aryl hydrocarbon or PPAR-alpha nuclear receptors.
Cell-free membrane assays with luminogenic substrates were used to
measure P450 or monoamine oxidase enzyme activities and their
inhibition by test compounds. IC50s from these bioluminescent assays
correlated well with conventional methods in terms of rank order and
absolute potency. Each bioluminescent assay relied on the light
generating reaction of firefly luciferase, coupling light output
with target activity. The assays were insensitive to interference
from fluorescent analytes and had low intrinsic background signals
giving them high levels of sensitivity and large dynamic ranges for
robust, high throughput applications. |
