Activity of dual SRC-ABL inhibitors highlights the role of BCR/ABL kinase dynamics in drug resistance

Abstract

Mutation in the ABL kinase domain is the principal mechanism of imatinib resistance in patients with chronic myelogenous leukemia. Many mutations favor active kinase conformations that preclude imatinib binding. Because the active forms of ABL and SRC resemble one another, we tested two dual SRC-ABL kinase inhibitors, AP23464 and PD166326, against 58 imatinib-resistant (IM R ) BCR͞ABL kinase variants. Both compounds potently inhibit most IM R variants, and in vitro drug selection demonstrates that active (AP23464) and open (PD166326) conformation-specific compounds are less susceptible to resistance than imatinib. Combinations of inhibitors suppressed essentially all resistance mutations, with the notable exception of T315I. Guided by mutagenesis studies and molecular modeling, we designed a series of AP23464 analogues to target T315I. The analogue AP23846 inhibited both native and T315I variants of BCR͞ABL with submicromolar potency but showed nonspecific cellular toxicity. Our data illustrate how conformational dynamics of the ABL kinase accounts for the activity of dual SRC-ABL inhibitors against IM R -mutants and provides a rationale for combining conformation specific inhibitors to suppress resistance.kinase inhibitors ͉ imatinib ͉ combination chemotherapy ͉ chronic myelogenous leukemia T he small molecule protein kinase inhibitors imatinib (1-5), gefitinib (6), and erlotinib (7, 8) are susceptible to resistance in patients because of amino acid substitutions in the target protein.Imatinib inhibits BCR͞ABL by stabilizing the kinase in a catalytically inactive conformation (9). Point mutations in the ABL kinase domain can thwart drug binding by direct steric hindrance or by destabilizing the inactive kinase conformation that is required for imatinib binding (2, 3). Consequently, developing drugs that target the open or active conformation of the kinase may prove effective in rescuing patients who develop imatinib resistance.Previously, we carried out an in vitro screen for imatinib resistance and identified a large number of mutant amino acid residues outside the active site that did not appear to act by direct steric hindrance of drug binding. Several of these residues were homologous to SRC residues known to play critical roles in maintaining an assembled, autoinhibited SRC kinase conformation (10-13), and some previously had been implicated by site-directed mutagenesis in ABL kinase regulation (14,15). We reasoned that these conformational, or allosteric, mutants exerted effects on drug binding by favoring adoption of the active kinase conformation. Using inferences from the mutagenesis studies, we proposed a model for the assembled ABL kinase that closely resembled the autoinhibited SRC structure (3). Crystallographic and biochemical data published alongside our mutagenesis report confirmed that ABL indeed was regulated in a SRC-like manner (16)(17)(18).The striking similarity between the catalytically active states of the SRC and ABL kinases prompted us to investigate whether SRC kinase inhibitors might...

Gene expression profiling of imatinib and PD166326-resistant CML cell lines identifies Fyn as a gene associated with resistance to BCR-ABL inhibitors

“…PD166326 is a nanomolar pyridopyrimidine inhibitor of ABL kinase effective against resistant BCR-ABL mutants. Conversely to imatinib, PD166326 also inhibits Src kinases and as such may exert additional effects on CML cell lines (38,39). IM-R and PD-R cells exhibited a drastic decrease in imatinib and PD166326 sensitivity as shown by cell metabolism and caspase assays.…”

Molecular Dynamics Simulations Show That Conformational Selection Governs the Binding Preferences of Imatinib for Several Tyrosine Kinases

“…With over 500 kinases in the human genome (3), specificity is hard to achieve (11), and new molecules are needed as resistance mutants appear (8,79). Imatinib, nilotinib, and other "type II" inhibitors act by stabilizing the inactive DFG-out conformation, seen in eight kinases so far (16,80) and, which probably exists in many others.…”

“…In Src and many other kinases, it is known that the DFG-out conformation is less stable than DFG-in, and this can explain at least part of the Src/Abl specificity of type II inhibitors like imatinib (33). Similarly, resistance screens have identified amino acids in Abl that reduce the effectiveness of imatinib and whose structure differs in the DFG-out and DFG-in states; they were postulated to act by destabilizing the DFG-out state, increasing ⌬G ii (8,33,37,38,79). However, no direct experimental proof or quantitative measurement of ⌬G ii has been obtained for any kinase.…”

Quantitative monitoring by polymerase colony assay of known mutations resistant to ABL kinase inhibitors

“…One can readily determine when mutations occur on a single DNA strand because they will appear on the same polony. Indeed, the simultaneous occurrence of multiple cooperating mutations in the same protein has been observed following in vitro mutagenesis and selection (Azam et al, 2006) and is beginning to be observed in clinical practice . Polony technology also allows for the quantification of BCR-ABL to ABL ratio and mutation haplotyping (Supplementary Figure), another example of its versatility.…”