Inexpensive High-Throughput Screening of Kinase Inhibitors Using One-Step Enzyme-Coupled Fluorescence Assay for ADP Detection

Abstract

Protein kinases are attractive targets for both biological research and drug development. Several assay kits, especially for the detection of adenosine diphosphate (ADP), which is universally produced by kinases, are commercially available for high-throughput screening (HTS) of kinase inhibitors, but their cost is quite high for large-scale screening. Here, we report a new enzyme-coupled fluorescence assay for ADP detection, which uses just 10 inexpensive, commercially available components. The assay protocol is very simple, requiring only the mixing of test solutions with ADP detection solution and reading the fluorescence intensity of resorufin produced by coupling reaction. To validate the assay, we focused on CDC2-like kinase 1 (CLK1), a dual-specificity kinase that plays an important role in alternative splicing, and we used the optimized assay to screen an in-house chemical library of about 215,000 compounds for CLK1 inhibitors. We identified and validated 12 potent inhibitors of CLK1, including a novel inhibitory scaffold. The results demonstrate that this assay platform is not only simple and cost-effective, but also sufficiently robust, showing good reproducibility and giving similar results to those obtained with the widely used ADP-Glo bioluminescent assay.

Temperature does matter—an additional dimension in kinase inhibitor development

“…Kinase and substrate were incubated at the indicated temperatures with 32 P‐γ‐ATP as described in Ref. [6]. A representative gel is shown.…”

“…However, recent work suggests an additional dimension that has been neglected in previous screening campaigns and validations: the impact of temperature, more specifically body temperature. In fact, common high‐throughput screening platforms perform a kinase reaction at room temperature or 30 °C (e.g., [4–6] also see http://www.kinase-screen.mrc.ac.uk/), which is obviously below human body temperature, that is, the environment where the inhibitor should eventually be used. Here we suggest that this discrepancy in temperature may contribute to false positives and false negatives in high‐throughput inhibitor screens and argue that temperature is an important variable that should be considered when screening for and validating kinase inhibitors.…”

The Academic Pill: How Academia Contributes to Curing Diseases

“…It supports academic and industrial researchers who want to screen chemical samples to find either chemical biological tools, drug leads, or agrochemicals. 6 The Drug Discovery Initiative has constructed a chemical library consisting of about 280,000 samples chosen primarily on druggability and structural diversity. The collection includes 63,000 samples deposited by industry since 2006.…”

“…Using phenotypic signatures for discovering the mode of action of compounds is a very active research field, and it is not surprising to see that all three analytical articles focus on that subject. The manuscripts of Starkuviene et al, 5 Imamura et al, 6 Colussi et al, 7 Close et al, 8 Siva et al, 9 and Wiseman et al 10 all describe novel model systems, assays, and technologies that allow the screening of large collections of molecules. This illustrates that academia is a rich source for novel assays as basic research is transformed into screens, leading to unexplored therapeutic avenues.…”

Identification of a New Inhibitor That Stabilizes Interleukin-2-Inducible T-Cell Kinase in Its Inactive Conformation

“…6 Targeting the inactive form of kinase offers advantages for achieving selectivity because of their structural diversification. 7 Traditional screening approaches for kinase inhibitors are commonly based on enzyme activity; 8,9 however, they do not provide information on the conformational state of the target. Hence, identifying those compounds from the primary screen hits that stabilize inactive conformations has provided an important way of achieving selectivity.…”