Mutation in the ABL kinase domain is the principal mechanism of imatinib resistance (IMR) in patients with chronic myelogenous leukemia (CML). The second generation BCR/ABL inhibitors, Nilotinib and Dasatinib, are effective in inhibiting essentially all IMR variants, but not the gatekeeper mutant T315I. Substitution of a bulky hydrophobic residue for the gatekeeper threonine not only causes steric blockade to the inhibitor but also stabilizes the active kinase conformation through a network of hydrophobic connections dubbed the hydrophobic-spine. In this study we describe the molecular mechanisms employed by the gatekeeper mutation to stabilize the active conformation, and demonstrate that these structural components can be targeted by the small molecule inhibitor compound #14, which efficiently inhibits native and gatekeeper mutant forms of the BCR/ABL kinase. Structural modeling and mutagenesis of residues constituting the spine suggests that compound #14 inhibits the kinase by disrupting the hydrophobic spine. Screening for drug resistance in vitro selected for clones having compound mutations involving both the P-loop and gatekeeper residues. Our studies provide structural guidance for the design of inhibitors against the gatekeeper mutant, and suggest that combination therapy may be required to prevent the emergence of compound resistance mutations.

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