Abstract 3728: A discovery platform for allosteric modulators of c-Abl kinase function

Abstract

Abstract The c-Abl protein-tyrosine kinase regulates intracellular signaling pathways controlling cell growth, adhesion, and responses to genotoxic stress. The kinase activity of c-Abl is repressed by intramolecular interactions involving its non-catalytic SH3 and SH2 domains. The SH3 domain interacts with a polyproline type II helix formed by the SH2-kinase linker, while the SH2 domain interacts with the C-lobe of kinase domain away from the active site. In addition, the myristoylated N-terminal cap region (Ncap) binds a hydrophobic pocket in the C-lobe of the kinase domain, clamping the SH3 and SH2 domains against the back of the kinase domain. Small molecules that allosterically regulate c-Abl kinase activity through its non-catalytic domains may represent selective probes of c-Abl function in diverse tumor types. In this study, we developed a chemical library screening assay designed to identify chemical modulators of c-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 purified protein consisting of the c-Abl Ncap, SH3 and SH2 domains plus the linker (N32L protein) and a short fluorescein-labeled probe peptide that binds to the SH3 domain. In assay development experiments, we found that the probe peptide binds to the recombinant c-Abl N32L protein in vitro, producing a robust FP signal that can be competed with an excess of unlabeled peptide. The FP signal is not observed with control N32L proteins bearing either an inactivating mutation in the SH3 domain or enhanced SH3:linker interaction. The assay was optimized with respect to probe peptide sequence, probe and N32L protein concentrations, DMSO tolerance, and FP signal stability over time. Pilot library screens were then performed with the NCI Diversity Set III and an FDA-approved drug library (2800 compounds total). Seven compounds were identified that specifically reduced the FP signal by at least three standard deviations from the untreated controls. Secondary assays showed that one of these hit compounds enhances c-Abl kinase activity in vitro, suggesting that it disrupts regulation of kinase activity through the non-catalytic region. These results show that screening assays based on the non-catalytic domains of c-Abl can identify small molecule regulators of kinase function. Citation Format: Prerna Grover, Thomas E. Smithgall. A discovery platform for allosteric modulators of c-Abl kinase function. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3728. doi:10.1158/1538-7445.AM2014-3728