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

David C. Smithson, Anang A. Shelat, Jeffrey Baldwin, Margaret A. Phillips, R. Kiplin Guy

Research output: Contribution to journalArticle

11 Scopus citations

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 ∼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).

Original languageEnglish (US)
Pages (from-to)175-185
Number of pages11
JournalAssay and Drug Development Technologies
Volume8
Issue number2
DOIs
StatePublished - Apr 1 2010

ASJC Scopus subject areas

  • Molecular Medicine
  • Drug Discovery

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