Configuration of a Scintillation Proximity Assay for the Activity Assessment of Recombinant Human Adenine Phosphoribosyltransferase

Abstract

Adenine phosphoribosyltransferase plays a role in purine salvage by catalyzing the direct conversion of adenine to adenosine monophosphate. The involvement of the purine salvage pathway in tumor proliferation and angiogenesis makes adenine phosphoribosyltransferase a potential target for oncology drug discovery. We have expressed and characterized recombinant, N-terminally His-tagged human adenine phosphoribosyltransferase. Two assay formats were assessed for use in a high throughput screen: a spectrophotometric-based enzyme-coupled assay system and a radiometric ionic capture scintillation proximity bead assay format. Ultimately, the scintillation proximity assay format was chosen because of automated screening compatibility limitations of the coupled assay. We describe here the biochemical characterization of adenine phosphoribosyltransferase and the development of a robust, homogeneous, 384-well assay suitable for high throughput screening.

Development and Validation of a Transcreener Assay for Detection of AMP- and GMP-Producing Enzymes

“…20 Transcreener FP assays are based on a competitive fluorescence polarization immunoassay (FPIA), in which the nucleotide analyte displaces a tracer from antibody resulting in a decreased polarization value. 1 The 2 detection reagents are an antibody and a tracer, comprised of the analyte nucleotide conjugated to a fluor.…”

Crystal structures of APRT from Francisella tularensis – an N–H···N hydrogen bond imparts adenine specificity in adenine phosporibosyltransferases

“…Further, fast-growing bacteria or cancer cells are proposed to use the salvage pathway, with its economical single-step production of nucleotides. In fact, human APRT has been considered as a target to block fast-growing cancer cells and to develop lead molecules for cancer chemotherapy [8]. APRTs from several pathogenic organisms have also been studied, considered as potential targets for chemotherapy [9][10][11][12][13][14][15].…”