Current In Vitro Kinase Assay Technologies: The Quest for a Universal Format

Abstract

The rapidly growing interest in kinases as drug targets has prompted the development of many kinase assay technologies. These technologies can be grouped into three categories: radiometric assays, phospho-antibody-dependent fluorescence/luminescence assays, and phospho-antibody-independent fluorescence/luminescence assays. This article will review some of the major kinase assay technologies on the market, with particular emphasis on the newest systems. We will describe the physical principles, the practical advantages and drawbacks, and the potential applications of these technologies in kinase drug discovery. Most of these technologies are suitable for HTS, but only a few can be utilized for kinetic and mechanistic studies. Significant progress towards development of generic assays, free of radioisotopes and custom reagents such as phospho-specific antibodies, has been made in recent years. However, due to various limitations of each format, none of these generic assay technologies can yet claim to be truly universal. Several factors, including the intended applications, cost, timeline, expertise, familiarity, and comfort level, should be considered prior to pursuing a particular kinase assay technology.

Design and evaluation of a real-time activity probe for focal adhesion kinase

“…Consequently, while radioactivity assays remain the "gold standard" for detailed mechanistic studies of protein kinases, there is a need for the development of assay formats that are compatible with high-throughput screening (HTS) for this important class of drug targets [11,12]. This has led to the development of a variety of assay formats for protein kinases [13]. These assays utilize various mechanisms to monitor kinase activity including production of ADP [14], protease sensitivity of phosphorylated peptides [15], time-resolved FRET [16,17], and the amplified luminescent proximity homogeneous assay format [18].…”

Targeted mass spectrometry: An emerging powerful approach to unblock the bottleneck in phosphoproteomics

“…Traditionally, protein phosphorylation has been investigated using various biochemical approaches that rely on the use of radioactivity ( 32 P), phospho-specific dyes or in vitro kinase assays [19][20][21]. Although the contribution of these types of studies is invaluable, they do not provide information about the precise site or sites that are modified within the phosphoprotein.…”

Human protein kinase inhibitor screening by capillary electrophoresis using transverse diffusion of laminar flow profiles for reactant mixing

“…Usually, fluorimetric, radiometric, antibody-dependent spectroscopic and antibody-independent spectroscopic assays are employed for kinase studies [2,3]. These methods require several after-reaction processing steps and are generally expensive.…”