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.

To realize the full potential of targeted protein kinase inhibitors for the treatment of cancer, it is important to address the emergence of drug resistance in treated patients. Mutant forms of BCR-ABL, KIT, and the EGF receptor (EGFR) have been found that confer resistance to the drugs imatinib, gefitinib, and erlotinib. The mutations weaken or prevent drug binding, and interestingly, one of the most common sites of mutation in all three kinases is a highly conserved ''gatekeeper'' threonine residue near the kinase active site. We have identified existing clinical compounds that bind and inhibit drug-resistant mutant variants of ABL, KIT, and EGFR. We found that the Aurora kinase inhibitor VX-680 and the p38 inhibitor BIRB-796 inhibit the imatinib-and BMS-354825-resistant ABL(T315I) kinase. The KIT͞FLT3 inhibitor SU-11248 potently inhibits the imatinib-resistant KIT(V559D͞T670I) kinase, consistent with the clinical efficacy of SU-11248 against imatinib-resistant gastrointestinal tumors, and the EGFR inhibitors EKB-569 and CI-1033, but not GW-572016 and ZD-6474, potently inhibit the gefitiniband erlotinib-resistant EGFR(L858R͞T790M) kinase. EKB-569 and CI-1033 are already in clinical trials, and our results suggest that they should be considered for testing in the treatment of gefitinib͞ erlotinib-resistant non-small cell lung cancer. The results highlight the strategy of screening existing clinical compounds against newly identified drug-resistant mutant variants to find compounds that may serve as starting points for the development of nextgeneration drugs, or that could be used directly to treat patients that have acquired resistance to first-generation targeted therapy. drug resistance ͉ gatekeeper mutation ͉ kinase inhibitor

The upstream protein kinases responsible for thousands of phosphorylation events in the phosphoproteome remain to be discovered. We developed a three-component chemical reaction which converts the transient non-covalent substrate-kinase complex into a covalently cross-linked product by utilizing a dialdehyde based cross-linker, 1. Unfortunately the reaction of 1 with a lysine in the kinase active site and an engineered cysteine on the substrate to form an isoindole cross-linked product could not be performed in the presence of competing cellular proteinsdue to non-specific side reactions. In order to more selectively target the cross-linker to protein kinases in cell lysates we replaced the weak, kinase-binding adenosine moiety of 1 with a potent protein kinase inhibitor scaffold. In addition, we replaced the o-phthaldialdehyde moiety in 1 with a less reactive thiophene-2,3-dicarboxaldehyde moiety. The combination of these two structural modifications provides for cross-linking of a cysteine containing substrate to its corresponding kinase in the presence of competing cellular proteins.