UNDERSTANDING ACTIVE ABL KINASE CONFORMATIONS: APPLICATION TO DISCOVERY OF SMALL MOLECULE ALLOSTERIC MODULATORS

[摘要] The c-Abl protein-tyrosine kinase regulates diverse cellular signaling pathways involved in cell growth, adhesion, and responses to genotoxic stress. Abl is well known in the context of Bcr-Abl, the active fusion tyrosine kinase, which causes chronic myelogenous leukemia (CML) and other leukemias. The tyrosine kinase activity of Abl is tightly regulated by auto-inhibitory interactions involving its non-catalytic SH3 and SH2 domains. Mutations that perturb these intramolecular interactions result in kinase activation. This study examined the effect of activating mutations on the biochemistry and solution structure of Abl core proteins. In an active myristic acid-binding pocket mutant (A356N), the relative positions of the regulatory N-cap, SH3 and SH2 domains were virtually identical to those of the assembled wild-type core despite differences in catalytic activity and thermal stability. In contrast, a dramatic structural rearrangement in an active gatekeeper mutant (T315I) was observed with the positions of the SH2 and SH3 domains reversed relative to wild-type. These results show that Abl kinases can adopt multiple conformations in solution and kinase activation does not necessarily require destabilization of the assembled core structure.Small molecules that allosterically regulate Abl kinase activity through its non-catalytic domains may represent selective probes of Abl function. I developed a screening assay to identify chemical modulators of Abl kinase activity that either disrupt or stabilize the regulatory interaction of the SH3 domain with the SH2-kinase linker. This fluorescence polarization (FP) assay is based on a recombinant Abl protein containing the regulatory domains (Ncap-SH3-SH2-linker, N32L) and a short fluorescein-labeled probe peptide that binds the SH3 domain. The probe peptide binds the recombinant Abl N32L protein in vitro producing a robust FP signal. Mutation of the SH3 binding site (W118A) or introduction of a high-affinity linker both resulted in loss of the FP signal. Pilot screens were performed with two chemical libraries (2800 compounds total), and thirteen compounds were found to specifically inhibit the FP signal. Secondary assays showed that one of these hit compounds enhances Abl kinase activity in vitro. These results show that screening assays based on the regulatory domains of Abl can identify allosteric modulators of kinase function.

[发布机构] the University of Pittsburgh