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.

Comparison of Bioluminescent Kinase Assays Using Substrate Depletion and Product Formation

“…The red-shifted fl uorescence limits spectroscopic interference by compounds and the ratiometric nature of fl uorescent-polarization measurements aides in minimizing artifacts due to liquid handling. 11,12 Further, this system shows good assay performance at low (~10%) conversion levels.…”

High-Throughput Screening for RecA Inhibitors Using a Transcreener Adenosine 5′-O-Diphosphate Assay

“…20 Moreover, the assays have proven to be suitable for high-throughput screening (HTS). 21 In the present study,…”

Regulators of G-Protein Signaling and Their Gα Substrates: Promises and Challenges in Their Use as Drug Discovery Targets

“…To detect RGS protein-accelerated GTPase activity without the use of radioactivity, a monoclonal antibody and fluorescent tracer system, previously developed by Bellbrook Labs for their Transcreener ADP assay (Kleman-Leyer et al, 2009), was adapted for selective immunodetection of GDP with a fluorescence polarization (FP) readout (Fig. 9).…”