Miles A. Fabian

A small molecule–kinase interaction map for clinical kinase inhibitors

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.

Tuning a Three-Component Reaction For Trapping Kinase Substrate Complexes

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 noncovalent 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 proteins due to nonspecific 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.

Arylcarboxyamino-substituted diaryl ureas as potent and selective FLT3 inhibitors

A series of diaryl ureas with an amide substitution at the 4-position was prepared and found to be potent and selective FLT3 inhibitors with good oral bioavailability and efficacy in a tumor xenograft model.

Reply to BIRB-796 is not an effective ABL(T315I) inhibitor

VOLUME 23 NUMBER 10 OCTOBER 2005 NATURE BIOTECHNOLOGY within a clinically achievable range, whereas a 100-fold difference would require levels in excess of 4 μM. Given the large possible range of values for enzyme inhibition and the clinical implications, we further evaluated BIRB-796. We initially tested the effect of BIRB-796 (0–5 μM) on the proliferation of Ba/F3 cells expressing either unmutated BCR-ABL or BCR-ABL with a kinase domain mutation associated with imatinib resistance. In line with the results of the binding assay described by Fabian et al. (Table 1), BIRB-796 was ineffective at all tested concentrations against cells expressing nonmutated BCRABL or mutants Y253F, E255K and M351T (data not shown). However, BIRB-796 also failed to inhibit proliferation of cells expressing BCR-ABL(T315I). Even when the BIRB-796 concentration range was extended to 20 μM for cells expressing BCR-ABL or BCR-ABL(T315I), the IC50 was not reached. As a control, we confirmed that BIRB-796 efficiently blocked the ability of p38α to phosphorylate MAPKAP-K2, an exclusive substrate of p38α, with an IC50 value of <2 nM (data not shown). In purified kinase assays, BIRB-796 exhibited a weak inhibitory effect on the catalytic activity of full-length ABL and ABL(T315I), reaching the IC50 value at an inhibitor concentration of 5.3 μM in both cases (Table 1). Given these findings, it is unlikely that BIRB-796 would be of clinical utility for patients harboring this mutation. The new method for mapping smallmolecule inhibitor-kinase interactions described by Fabian et al. appears to be useful both for quickly evaluating kinase specificity and as an initial screen to identify unanticipated inhibitor targets. However, given the wide range of differences between binding measured in this assay and enzymatic assays, inhibitor candidates with acceptable binding profiles must then be subjected to assays that directly measure inhibition rather than binding.