Optimization of a non-radioactive high-throughput assay for decarboxylase enzymes

Assay Drug Dev Technol. 2010 Apr;8(2):175-85. doi: 10.1089/adt.2009.0249.


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

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Animals
  • Bicarbonates / analysis
  • Carboxy-Lyases / analysis*
  • Carboxy-Lyases / antagonists & inhibitors
  • Carboxy-Lyases / isolation & purification
  • Data Interpretation, Statistical
  • Drug Evaluation, Preclinical / methods
  • Enzyme Assays
  • Enzyme Inhibitors / pharmacology
  • Malate Dehydrogenase / analysis
  • Ornithine Decarboxylase / analysis
  • Ornithine Decarboxylase / metabolism
  • Ornithine Decarboxylase Inhibitors
  • Phosphoenolpyruvate Carboxylase / analysis
  • ROC Curve
  • Trypanosoma brucei brucei / enzymology


  • Bicarbonates
  • Enzyme Inhibitors
  • Ornithine Decarboxylase Inhibitors
  • Malate Dehydrogenase
  • Carboxy-Lyases
  • Ornithine Decarboxylase
  • Phosphoenolpyruvate Carboxylase