Kinase inhibitors are a new class of therapeutics with a propensity to inhibit multiple targets. The biological consequences of multi-kinase activity are poorly defined, and an important step toward understanding the relationship between selectivity, efficacy and safety is the exploration of how inhibitors interact with the human kinome. We present interaction maps for 38 kinase inhibitors across a panel of 317 kinases representing >50% of the predicted human protein kinome. The data constitute the most comprehensive study of kinase inhibitor selectivity to date and reveal a wide diversity of interaction patterns. To enable a global analysis of the results, we introduce the concept of a selectivity score as a general tool to quantify and differentiate the observed interaction patterns. We further investigate the impact of panel size and find that small assay panels do not provide a robust measure of selectivity.

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.

Activating mutations in the receptor tyrosine kinase FLT3 are present in up to approximately 30% of acute myeloid leukemia (AML) patients, implicating FLT3 as a driver of the disease and therefore as a target for therapy. We report the characterization of AC220, a second-generation FLT3 inhibitor, and a comparison of AC220 with the first-generation FLT3 inhibitors CEP-701, MLN-518, PKC-412, sorafenib, and sunitinib. AC220 exhibits low nanomolar potency in biochemical and cellular assays and exceptional kinase selectivity, and in animal models is efficacious at doses as low as 1 mg/kg given orally once daily. The data reveal that the combination of excellent potency, selectivity, and pharmacokinetic properties is unique to AC220, which therefore is the first drug candidate with a profile that matches the characteristics desirable for a clinical FLT3 inhibitor. (Blood. 2009; 114:2984-2992) IntroductionThe presence of genetic changes in cancer cells that lead to dysregulated activation of kinases frequently signals that the activated kinase is a contributing driver of disease, 1-4 and inhibitors of activated kinases can have a dramatic impact on disease progression in patients with these genetic alterations. 5,6 To clearly define the role of the dysregulated kinase, and to determine whether inhibition of the mutant kinase is sufficient to induce a therapeutic benefit, requires drugs capable of selectively, potently, and preferably sustainably inhibiting the activated kinase in patients.Activating mutations in the FLT3 receptor tyrosine kinase have been identified in up to 30% of acute myeloid leukemia (AML) patients. 7,8 The most common class of mutation is internal tandem duplications (ITDs) in the juxtamembrane domain 7,9 that lead to constitutive, ligand-independent activation of the kinase. 7,10 The prognosis for patients with FLT3-ITD mutations is significantly worse than that for patients with wild-type FLT3 when treated with standard therapy. [7][8][9][11][12][13][14][15][16] The presence of activating FLT3 mutations and the correlation of FLT3 activation with a poor prognosis strongly suggest that FLT3 is a driver of disease in AML, at least in patients with FLT3-ITD mutations. Several small molecule kinase inhibitors with activity against FLT3 have been evaluated in AML patients, including CEP-701 (lestaurtinib), PKC-412 (midostaurin), MLN-518 (tandutinib; previously known as CT-53518), sunitinib (SU-11248), sorafenib , and KW-2449. The compounds inhibit FLT3 in cellular assays and are efficacious in mouse models of FLT3-ITD AML. [17][18][19][20][21][22] In phase 1 and phase 2 clinical trials, conducted primarily in relapsed or refractory AML patients, responses were consistently observed with each of these drugs, 21,[23][24][25][26][27][28][29][30][31] however, responses generally were relatively limited and not durable. 21,[23][24][25]30 The studies did reveal a relationship between the likelihood of observing a clinical response and the pharmacokinetics/pharmacodynamics of FLT3 inhibition, and highlight...

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