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 Citations (Scopus)

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

Fingerprint

Carboxy-Lyases
Enzyme Assays
Trypanosoma brucei brucei
Ornithine Decarboxylase
Bicarbonates
Atmosphere
ROC Curve
Libraries
Area Under Curve
Nitrogen
Confidence Intervals

ASJC Scopus subject areas

  • Drug Discovery
  • Molecular Medicine

Cite this

Optimization of a non-radioactive high-throughput assay for decarboxylase enzymes. / Smithson, David C.; Shelat, Anang A.; Baldwin, Jeffrey; Phillips, Margaret A.; Guy, R. Kiplin.

In: Assay and Drug Development Technologies, Vol. 8, No. 2, 01.04.2010, p. 175-185.

Research output: Contribution to journalArticle

Smithson, David C. ; Shelat, Anang A. ; Baldwin, Jeffrey ; Phillips, Margaret A. ; Guy, R. Kiplin. / Optimization of a non-radioactive high-throughput assay for decarboxylase enzymes. In: Assay and Drug Development Technologies. 2010 ; Vol. 8, No. 2. pp. 175-185.
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