Profiling used in lead optimization and drug discovery
Protein kinase profiling assays: a technology review

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Protein kinases have become one of the most intensively pursued classes of drug targets for many diseases such as cancers and inflammatory diseases. Kinase profiling work seeks to understand general selectivity trends of lead compounds across the kinome, which help with target selection, compound prioritization, and potential implications in toxicity. Under the current drug discovery process, screening of compounds against comprehensive panels of kinases and their mutants has become the standard approach. Many screening assays and technologies which are compatible for high-throughput screening (HTS) against kinases have been extensively pursued and developed.

Introduction

Protein kinases are one of the largest families of evolutionarily related proteins and comprise one of the most abundant gene families in humans. The human kinome comprises 518 protein kinases and approximately 20 lipid kinases [1]. Protein kinases act on proteins, phosphorylating them on their serine, threonine, tyrosine, or histidine residues. Phosphorylation can modify a protein functional activity in cells in many ways [2], [3], [4]. During the last couple of decades, much clinical evidence has demonstrated that the protein kinases represent some of the most effective therapeutic targets in various types of cancer [5]. Over 20 kinase inhibitors for the treatment of cancers and inflammatory diseases have been approved by the FDA, beginning in 2001 with the approval of imatinib [6]. The market for kinase inhibitors is expected to continue to grow, with worldwide sales forecasted to reach approximately $20 billion in 2015 [7]. In addition, knowledge of the off-target effects of a kinase inhibitor can be important to understand their biological mechanism particularly for non-selective compounds, to anticipate their potential toxicities, as well as to discover unexpected activities which could lead to different chemical design and possibly novel therapeutic opportunities [8].

Due to the large size of the protein kinase family, many screening technologies which are compatible for high-throughput screening (HTS) against comprehensive panels of kinases and their mutants have been extensively pursued and developed [9], [10], [11]. However, the plethora of assay formats also poses a challenge to decide which assay platform or technology will be the best fit. Various factors such as the cost, efficiency, and how convenient the assay is to use, as well as low rates of false positive/negative results should be considered as well. In the sections below, we will discuss the most commonly used kinase assay methods along with their pros and cons in general drug screening applications.

Section snippets

Kinase functional assays

Kinase functional assays, also known as activity-based assays or enzymatic assays, directly or indirectly quantify the catalytic product production (i.e., the phosphorylated substrate, ADP or γ-phosphate) [10], [11]. In a survey of drug discovery labs and companies worldwide in 2013, functional assays were the most preferred format when compared to binding-based and cellular assays [12]. Functional assay platforms include radiometric, fluorescence-based, luminescence-based, and mobility shift

Kinase binding assays

Binding assays quantitatively measure the binding of small molecules to the kinase protein, rather than measuring catalytic product. The binding assays are valuable in determining the target engagement and binding affinity. Unlike functional assays, the kinase binding assay can be performed with either active or non-activated kinase preparations. On the other hand, because binding assays are usually performed without ATP or substrate, they are generally unable to detect substrate-specific

Conclusion

Kinase profiling seeks to determine the specificity of a compound against a large panel of diverse kinases. As such, a profiling assay format should be applicable to all kinases within the panel, and immune from interferences both from detections and compounds. By comparison of the assay formats discussed above, the radioisotope-based filtration binding assay stands out as one of the most favorable choices for kinase profiling activity assays because it can universally apply to all protein

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