Imatinib Alters Agonists-mediated Cytoskeletal Biomechanics in Lung Endothelium.

The endothelium serves as a size-selective barrier and tightly controls the fluid exchange from the circulation to the surrounding tissues. In this study, a multiplexed microscopy characterization is developed to study the spatio-temporal effects of Abl kinases on endothelial cytoskeletal structure using AFM, SEM, and immunofluorescence. Sphingosine 1-phosphate (S1P) produces significant endothelial barrier enhancement by means of peripheral actin rearrangement. However, Abl kinase inhibition by imatinib reduces rapid redistribution of the important cytoskeletal proteins to the periphery and their association with the cortical actin ring. Herein, it moderates the thickness of the cortical actin ring, and diminishes the increase in elastic modulus at the periphery and cytoplasm. These findings demonstrate that imatinib attenuates multiple cytoskeletal changes associated with S1P-mediated endothelial barrier enhancement and suggest a novel role for Abl kinases in mediating these S1P effects. These observations bridge the gap between molecule dynamics, structure complexity and function connectivity across varied length-scales to improve our understanding on human pulmonary endothelial barrier regulation. Moreover, our study suggests a framework for understanding form-function relationships in other biomechanical subsystems, wherein complex hierarchical organization programmed from the molecular scale to the cellular and tissue levels has an intimate relationship to the overall physiological function.

Fluorescence Polarization Screening Assays for Small Molecule Allosteric Modulators of ABL Kinase Function.

The ABL protein-tyrosine kinase regulates intracellular signaling pathways controlling diverse cellular processes and contributes to several forms of cancer. The kinase activity of ABL is repressed by intramolecular interactions involving its regulatory Ncap, SH3 and SH2 domains. Small molecules that allosterically regulate ABL kinase activity through its non-catalytic domains may represent selective probes of ABL function. Here we report a screening assay for chemical modulators of ABL kinase activity that target the regulatory interaction of the SH3 domain with the SH2-kinase linker. This fluorescence polarization (FP) assay is based on a purified recombinant ABL protein consisting of the N-cap, SH3 and SH2 domains plus the SH2-kinase 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 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. A pilot screen of 1200 FDA-approved drugs identified four compounds 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, the antithrombotic drug dipyridamole, enhances ABL kinase activity in vitro to a greater extent than the previously described ABL agonist, DPH. Docking studies predicted that this compound binds to a pocket formed at the interface of the SH3 domain and the linker, suggesting that it activates ABL by disrupting this regulatory interaction. These results show that screening assays based on the non-catalytic domains of ABL can identify allosteric small molecule regulators of kinase function, providing a new approach to selective drug discovery for this important kinase system.

Assay development and high-throughput screening of small molecular c-Abl kinase activators.

A 2-step kinase assay was developed and used in a high-throughput screen (HTS) of more than 1 million compounds in an effort to identify c-Abl tyrosine kinase activators. This assay employed a 2-step phosphorylation reaction: in the first step, purified recombinant c-Abl was activated by incubating with compound in the presence of adenosine triphosphate (ATP). In the second step, the TAMRA-labeled IMAP Abltide substrate was added to allow phosphorylation of the substrate to occur. The assay was calibrated such that inactive c-Abl protein was activated by ATP alone to a degree that it not only demonstrated a measurable c-Abl activity but also maintained a robust assay window for screening. The screen resulted in 8624 primary hits with >30% response. Further analysis showed that 1024 had EC(50) <10 µM with a max % response of >50%. These hits were structurally and chemically diverse with possibly different mechanisms for activating c-Abl. In addition, selective hits were shown to be cell permeable and were able to induce c-Abl activation as determined by In-Cell Western (ICW) analysis of HEK-MSRII cells transduced with BacMam virus expressing full-length c-Abl.

Binding or bending: distinction of allosteric Abl kinase agonists from antagonists by an NMR-based conformational assay.

Allosteric inhibitors of Bcr-Abl have emerged as a novel therapeutic option for the treatment of CML. Using fragment-based screening, a search for novel Abl inhibitors that bind to the myristate pocket was carried out. Here we show that not all myristate ligands are functional inhibitors, but that the conformational state of C-terminal helix_I is a structural determinant for functional activity. We present an NMR-based conformational assay to monitor the conformation of this crucial helix_I and show that myristate ligands that bend helix_I are functional antagonists, whereas ligands that bind to the myristate pocket but do not induce this conformational change are kinase agonists. Activation of c-Abl by allosteric agonists has been confirmed in a biochemical assay.