title: Characterization and optimization of a red-shifted fluorescence polarization ADP detection assay.
abstract: ATP depletion and ADP formation are generic detection methods used for the identification of kinase and other ATP-utilizing enzyme inhibitors in high-throughput screening campaigns. However, the most widely used nucleotide detection approaches require high ATP consumption rates or involve the use of coupling enzymes, which can complicate the selection of lead compounds. As an alternative, we have developed the Transcreener (BellBrook Labs, Madison, WI) platform, which relies on the direct immunodetection of nucleotides. Here we describe the development of antibodies with >100-fold selectivity for ADP versus ATP, which enable robust detection of initial velocity rates (Z' > 0.7 at 10% substrate consumption) at ATP concentrations ranging from 0.1 microM to 1,000 microM in a competitive fluorescence polarization (FP) immunoassay. Competitive binding experiments indicate similar affinities for other nucleotide diphosphates, including 2' -deoxy ADP, GDP, and UDP. The antibody-tracer complex and the red-shifted, ratiometric FP signal are stable for at least 24 h at room temperature, providing suitable conditions for high-throughput screening. A method for calculating a kinase ATP Km with this FP immunoassay is also presented. The Transcreener ADP assay provides a simple, generic assay platform for inhibitor screening and selectivity profiling that can be used for any ADP-generating enzyme.
authors.affiliation: BellBrook Labs, 5500 Nobel Drive Suite 250, Madison, WI 53711, USA. karen.kleman@bellbrooklabs.com
authors.forename: Karen M
authors.initials: KM
authors.lastname: Kleman-Leyer
authors.forename: Tony A
authors.initials: TA
authors.lastname: Klink
authors.forename: Andrew L
authors.initials: AL
authors.lastname: Kopp
authors.forename: Thane A
authors.initials: TA
authors.lastname: Westermeyer
authors.forename: Mark D
authors.initials: MD
authors.lastname: Koeff
authors.forename: Brad R
authors.initials: BR
authors.lastname: Larson
authors.forename: Tracy J
authors.initials: TJ
authors.lastname: Worzella
authors.forename: Cori A
authors.initials: CA
authors.lastname: Pinchard
authors.forename: Sebastianus A T
authors.initials: SA
authors.lastname: van de Kar
authors.forename: Guido J R
authors.initials: GJ
authors.lastname: Zaman
authors.forename: Jorrit J
authors.initials: JJ
authors.lastname: Hornberg
authors.forename: Robert G
authors.initials: RG
authors.lastname: Lowery
mesh_terms: D000244:Adenosine Diphosphate / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D002021:Buffers; D002851:Chromatography, High Pressure Liquid; D016231:Fluorescence Polarization Immunoassay / Q000379:methods*; D007202:Indicators and Reagents; D007700:Kinetics; D019151:Peptide Library; D011494:Protein Kinases / Q000378:metabolism; D012015:Reference Standards
chemical_list: D002021:Buffers; D007202:Indicators and Reagents; D019151:Peptide Library; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D011494:Protein Kinases