A small molecule–kinase interaction map for clinical kinase inhibitors

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Kinase inhibitors show great promise as a new class of therapeutics. Here we describe an efficient way to determine kinase inhibitor specificity by measuring binding of small molecules to the ATP site of kinases. We have profiled 20 kinase inhibitors, including 16 that are approved drugs or in clinical development, against a panel of 119 protein kinases. We find that specificity varies widely and is not strongly correlated with chemical structure or the identity of the intended target. Many novel interactions were identified, including tight binding of the p38 inhibitor BIRB-796 to an imatinib-resistant variant of the ABL kinase, and binding of imatinib to the SRC-family kinase LCK. We also show that mutations in the epidermal growth factor receptor (EGFR) found in gefitinib-responsive patients do not affect the binding affinity of gefitinib or erlotinib. Our results represent a systematic small molecule-protein interaction map for clinical compounds across a large number of related proteins.

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Figure 1: Competition binding assay for measuring the interaction between unlinked, unmodified ('free') small molecules and kinases.
Figure 2: Panel of binding assays for 113 different protein kinases.
Figure 3: Specificity profiles of clinical kinase inhibitors.
Figure 4: Distribution of binding constants.
Figure 5: Hierarchical cluster analysis of specificity profiles.
Figure 6: Relative binding affinities of EGFR inhibitors for wild type and mutant forms of EGFR.


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We thank Tony Hunter, Nicholas Lydon and Webster Cavenee for a critical reading of the manuscript and helpful discussions, Dan Lockhart for writing software tools to facilitate data analysis, David Austin for helpful suggestions regarding compound synthesis and Nicholas Olney and Victor Perez for expert technical assistance.

Author information

Correspondence to Patrick P Zarrinkar or David J Lockhart.

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Competing interests

All authors are current or former employees of Ambit Biosciences.

Supplementary information

Supplementary Table 1

Kinase inhibitors for which specificity profiles were determined (PDF 32 kb)

Supplementary Table 2

Comparison of binding constants measured in the competition binding assays to published results. (PDF 19 kb)

Supplementary Table 3

Comparison of the results of cell-based assays and binding assays for FLT3 and EGFR inhibitors. (PDF 9 kb)

Supplementary Table 4

Complete quantitative results of screening twenty kinase inhibitors against 119 protein kinases. (PDF 28 kb)

Supplementary Table 5

Binding constants for eight small molecules binding to ten EGFR variants. Binding constant values are in nanomolar. (PDF 10 kb)

Supplementary Table 6

List of clone type used for each kinase assay. Domain clones include the complete kinase catalytic domain along with flanking sequences. (PDF 18 kb)

Supplementary Notes (PDF 28 kb)

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