Measuring and interpreting the selectivity of protein kinase inhibitors

Abstract

Protein kinase inhibitors are a well-established class of clinically useful drugs, particularly for the treatment of cancer. Achieving inhibitor selectivity for particular protein kinases often remains a significant challenge in the development of new small molecules as drugs or as tools for chemical biology research. This review summarises the methodologies available for measuring kinase inhibitor selectivity, both in vitro and in cells. The interpretation of kinase inhibitor selectivity data is discussed, particularly with reference to the structural biology of the protein targets. Measurement and prediction of kinase inhibitor selectivity will be important for the development of new multi-targeted kinase inhibitors.

Fig. 1

X-ray crystal structure of CDK2 with ATP (black) bound with key structural motifs highlighted. Adapted from PDB: 1HCK

Fig. 2

Traxler model showing binding site for ATP in protein kinases [136]. Grey shading indicates non-conserved regions. GK gatekeeper residue

Fig. 3

X-ray crystal structure of dasatinib in Abl (Adapted from PDB: 2GQG)

Fig. 4

Pharmacophore model of inactive kinase conformation with sorafenib bound. Adapted from [94] and PDB: 1UWH. GK gatekeeper residue

Fig. 5

X-ray crystal structure of sorafenib in b-Raf (Adapted from PDB: 1UWH)

Fig. 6

X-ray crystal structures; a Allosteric MEK inhibitor bound in a pocket adjacent to the ATP binding site (Adapted from PDB: 1S9J); b Allosteric Chk1 inhibitor bound at a site remote from ATP binding site (Adapted from PDB: 3F9N)

Fig. 7

A schematic to summarise the various methods for detecting inhibition of the phosphorylation activity of kinases relevant to selectivity profiling

Fig. 8

Summary of various methods for detecting the binding of inhibitors to kinases. a Displacement of labelled probe compounds; b phage display of kinase domains with competitive binding to inhibitors or immobilised probe ligands; c affinity chromatography using immobilised inhibitor

Fig. 9

Schematic showing measurement of inhibitor selectivity using a a microarray of immobilised kinases; b a microarray of immobilised substrate peptides; c the yeast-3-hybrid proteomic method (AD activation domain, BD DNA binding domain); d tandem MS detection of altered binding of cellular kinase proteins to an affinity matrix following treatment with an inhibitor; e tandem MS detection of competitive binding to kinases using a reactive chemical label

Fig. 10

Examples of correlation (a) and non-correlation (b, c) of biochemical and cellular inhibitor selectivity between two kinases

Fig. 11

Selectivity profiles of three low molecular weight compounds (3, 4, 5) from one chemical series and their corresponding Gini co-efficients (G) and S_(40%) values. Percentages shown are percentage inhibition at 30 μM. The kinase dendrogram was adapted and reproduced with permission from Cell Signaling Technology, Inc. (www.cellsignal.com). With sponsorship by Cell Signaling Technology and Sugen, the figure was originally presented as a poster in Science to accompany the first analysis of the complete human kinome [100]