Abstract
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).
Publication types
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Research Support, N.I.H., Extramural
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Research Support, Non-U.S. Gov't
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Research Support, U.S. Gov't, Non-P.H.S.
MeSH terms
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Animals
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Bicarbonates / analysis
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Carboxy-Lyases / analysis*
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Carboxy-Lyases / antagonists & inhibitors
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Carboxy-Lyases / isolation & purification
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Data Interpretation, Statistical
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Drug Evaluation, Preclinical / methods
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Enzyme Assays
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Enzyme Inhibitors / pharmacology
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Malate Dehydrogenase / analysis
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Ornithine Decarboxylase / analysis
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Ornithine Decarboxylase / metabolism
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Ornithine Decarboxylase Inhibitors
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Phosphoenolpyruvate Carboxylase / analysis
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ROC Curve
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Trypanosoma brucei brucei / enzymology
Substances
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Bicarbonates
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Enzyme Inhibitors
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Ornithine Decarboxylase Inhibitors
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Malate Dehydrogenase
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Carboxy-Lyases
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Ornithine Decarboxylase
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Phosphoenolpyruvate Carboxylase