Characterization and optimization of a red-shifted fluorescence polarization ADP detection assay.
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.
Kleman-Leyer
Karen M
KM
BellBrook Labs, 5500 Nobel Drive Suite 250, Madison, WI 53711, USA. karen.kleman@bellbrooklabs.com
Klink
Tony A
TA
Kopp
Andrew L
AL
Westermeyer
Thane A
TA
Koeff
Mark D
MD
Larson
Brad R
BR
Worzella
Tracy J
TJ
Pinchard
Cori A
CA
van de Kar
Sebastianus A T
SA
Zaman
Guido J R
GJ
Hornberg
Jorrit J
JJ
Lowery
Robert G
RG
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
D002021:Buffers; D007202:Indicators and Reagents; D019151:Peptide Library; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D011494:Protein Kinases