TY - JOUR
T1 - Optimization of a non-radioactive high-throughput assay for decarboxylase enzymes
AU - Smithson, David C.
AU - Shelat, Anang A.
AU - Baldwin, Jeffrey
AU - Phillips, Margaret A.
AU - Guy, R. Kiplin
PY - 2010/4/1
Y1 - 2010/4/1
N2 - 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 ∼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).
AB - 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 ∼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).
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U2 - 10.1089/adt.2009.0249
DO - 10.1089/adt.2009.0249
M3 - Article
C2 - 20085486
AN - SCOPUS:77952076850
SN - 1540-658X
VL - 8
SP - 175
EP - 185
JO - Assay and Drug Development Technologies
JF - Assay and Drug Development Technologies
IS - 2
ER -