Bioluminescent assays for ADMET.
Bioluminescent assays couple a limiting component of a luciferase-catalyzed photon-emitting reaction to a variable parameter of interest, while holding the other components constant or non-limiting. In this way light output varies with the parameter of interest. This review describes three bioluminescent assay types that use firefly luciferase to measure properties of drugs and other xenobiotics which affect their absorption, distribution, metabolism, elimination and toxicity. First, levels of the luciferase enzyme itself are measured in gene reporter assays that place a luciferase cDNA under the control of regulatory sequences from ADMET-related genes. This approach identifies activators of nuclear receptors that regulate expression of genes encoding drug-metabolizing enzymes and drug transporters. Second, drug effects on enzyme activities are monitored with luminogenic probe substrates that are inactive derivatives of the luciferase substrate luciferin. The enzymes of interest convert the substrates to free luciferin, which is detected in a second reaction with luciferase. This approach is used with the drug-metabolizing CYP and monoamine oxidase enzymes, apoptosis-associated caspase proteases, a marker protease for non-viable cells and with glutathione-S-transferase to measure glutathione levels in cell lysates. Third, ATP concentration is monitored as a marker of cell viability or cell death and as a way of identifying substrates for the ATP-dependent drug transporter, P-glycoprotein. Luciferase activity is measured in the presence of a sample that supplies the requisite luciferase substrate, ATP, so that light output varies with ATP concentration. The bioluminescent ADMET assays are rapid and sensitive, amenable to automated high-throughput applications and offer significant advantages over alternative methods.
Cali
James J
JJ
Promega Corp., 2800 Woods Hollow Road, Madison, WI 53711, USA. jim.cali@promega.com
Niles
Andrew
A
Valley
Michael P
MP
O'Brien
Martha A
MA
Riss
Terry L
TL
Shultz
John
J
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D020169:Caspases / Q000737:chemistry / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D005978:Glutathione / Q000378:metabolism; D006801:Humans; D008156:Luciferases / Q000032:analysis / Q000378:metabolism; D049449:Luminescence; D006899:Mixed Function Oxygenases / Q000032:analysis / Q000378:metabolism; D004364:Pharmaceutical Preparations / Q000378:metabolism*; D010599:Pharmacokinetics
D004364:Pharmaceutical Preparations; D000255:Adenosine Triphosphate; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D008156:Luciferases; D020169:Caspases; D000251:Adenosine Triphosphatases; D005978:Glutathione