Optimization of a non-radioactive high-throughput assay for decarboxylase enzymes.
Herein, we describe the optimization of a linked enzyme assay suitable for high-throughput screening of decarboxylases, a target family whose activity has historically been difficult to quantify. Our approach uses a commercially available bicarbonate detection reagent to measure decarboxylase activity. The assay is performed in a fully enclosed automated screening system under inert nitrogen atmosphere to minimize perturbation by exogenous CO2. Receiver operating characteristic (ROC) analysis following a pilot screen of a small library of approximately 3,600 unique molecules for inhibitors of Trypanosoma brucei ornithine decarboxylase quantitatively demonstrates that the assay has excellent discriminatory power (area under the curve = 0.90 with 95% confidence interval between 0.82 and 0.97).
Smithson
David C
DC
Graduate Program in Chemistry and Chemical Biology, University of California, San Francisco, California, USA.
Shelat
Anang A
AA
Baldwin
Jeffrey
J
Phillips
Margaret A
MA
Guy
R Kiplin
RK
D000818:Animals; D001639:Bicarbonates / Q000032:analysis; D002262:Carboxy-Lyases / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000302:isolation & purification; D003627:Data Interpretation, Statistical; D004353:Drug Evaluation, Preclinical / Q000379:methods; D057075:Enzyme Assays; D004791:Enzyme Inhibitors / Q000494:pharmacology; D008291:Malate Dehydrogenase / Q000032:analysis; D009955:Ornithine Decarboxylase / Q000032:analysis / Q000378:metabolism; D065108:Ornithine Decarboxylase Inhibitors; D010730:Phosphoenolpyruvate Carboxylase / Q000032:analysis; D012372:ROC Curve; D014346:Trypanosoma brucei brucei / Q000201:enzymology
D001639:Bicarbonates; D004791:Enzyme Inhibitors; D065108:Ornithine Decarboxylase Inhibitors; D008291:Malate Dehydrogenase; D002262:Carboxy-Lyases; D009955:Ornithine Decarboxylase; D010730:Phosphoenolpyruvate Carboxylase