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. 2013 Mar:Chapter 2:Unit2.9.
doi: 10.1002/0471141755.ph0209s60.

The human kinome and kinase inhibition

Krisna C Duong-Ly  1 , Jeffrey R Peterson
Affiliations

The human kinome and kinase inhibition

Krisna C Duong-Ly et al. Curr Protoc Pharmacol. 2013 Mar.

Abstract

Protein and lipid kinases play key regulatory roles in a number of biological processes. Unsurprisingly, activating mutations in kinases have been linked to a number of disorders and diseases, most notably cancers. Thus, kinases have emerged as promising clinical targets. There are more than 500 human protein kinases and about 20 lipid kinases. Most protein kinases share a highly conserved domain, the eukaryotic protein kinase (ePK) domain, which contains the ATP and substrate-binding sites. Many inhibitors in clinical use bind to the highly conserved ATP binding site. For this reason, many kinase inhibitors are not exclusively selective for their intended targets. Furthermore, despite the current interest in kinase inhibitors, very few kinases implicated in disease have validated inhibitors. This unit describes the human kinome, ePK structure, and types of kinase inhibitors, focusing on methods to identify potent and selective kinase inhibitors.

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Figures

Figure 1
Figure 1. The human kinome
The human kinome consists of lipid and protein kinases. Human ePKs are classified into groups based on sequence similarity in the kinase domain (Manning et al., 2002). For kinase group abbreviations, see text. The ePK protein kinome tree was prepared using the Reaction Biology Corporation Kinome Activity Mapper (www.reactionbiology.com) and was adapted from and reproduced courtesy of Cell Signaling Technology, Inc. (www.cellsignal.com) based on Manning et al.
Figure 2
Figure 2. The eukaryotic protein kinase domain
(A) Top: The conformation of the inactive (PDB 1IRK) and active (PDB 1IR3) states of the kinase domain in IR. Elements of special significance are highlighted in color: C-helix (yellow), P-loop (red), hinge region (orange), catalytic loop (blue), activation loop (purple). The dashed line indicates the salt bridge between Glu1074 of the C-helix and Lys1030. Bottom: A close-up view of the surface of the ATP binding pocket for active and inactive IR. The binding of an ATP analog, AMP-PNP, is shown in green sticks. For ease of visualization, the conformation of AMP-PNP in active IR was docked into inactive IR where it clashes with the residues of the C-lobe. (B) Pockets adjacent to ATP that may also be exploited for inhibitor development.
Figure 3
Figure 3. Structures of inhibitors discussed in this review
Figure 4
Figure 4. Assay formats for measuring kinase-inhibitor interactions
(A) Melting temperature assay. In this test the shift in the melting temperature (ΔTm) correlates with the strength of the kinase-inhibitor interaction. In this example, inhibitor B interacts more potently with the kinase than inhibitor A since ΔTm, B is greater than ΔTm, A. (B) Competition binding assay. A ligand that binds the kinase is affixed on a solid support. Free inhibitor is passed over the solid support and competes with binding to the kinase, causing the enzyme to be “eluted” from the solid support. The concentration of the eluted kinase is measured and is an indicator of inhibitor binding. (C) Electrophoretic mobility assay. A polymer is conjugated to a phosphospecific antibody for the phosphorylation site of interest. Phosphorylated substrate binds the polymer and therefore migrates slower than unphosphorylated substrate that is not bound to the polymer. Visualization is possible if the substrate is conjugated to a fluorescent probe (indicated by the star). (D) ATP detection assay. The amount of unhydrolyzed ATP following a kinase reaction is assayed by coupling the reaction to the luciferase reaction. Luciferase requires ATP to oxidize luciferin to form oxyluciferin and to emit light. (E) Proximity assay. The substrate is conjugated to a fluorescent donor (star). The products of a kinase reaction utilizing this substrate are incubated with an antibody conjugated to a fluorescent acceptor (filled hexagon). Phosphorylated substrates bind the antibody and result in FRET (indicated by arrows and the open hexagons).
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