| Popis: |
Due to their central role in normal cellular physiology, the activity of protein kinases is tightly regulated and their aberrant activation can lead to cancer. Chronic myelogenous leukemia (CML) is a blood cancer characterized by unregulated growth of myeloid cells caused by a fusion protein, Bcr-Abl, a constitutively active formof the Abelson tyrosine kinase (Abl). While treatment with drugs targeting the ATP binding pocket of Abl leads to durable therapeutic responses, the development of drug resistance remains a major clinical problem. Targeting additional sites on kinases outside the conserved ATP pocket is thought to be a promising strategy to overcome drug resistance. We hypothesized that protein-interacting domains such as SH2 act as allosteric regulator of kinase activity and conformation. This project aimed at understanding the molecular mechanisms underlying regulation of Abl kinases via the SH2-kinase allosteric interface as well as developing a screening strategy for small-molecules disrupters of this interface. I used recombinant Abl tyrosine kinases and conformation-specific kinase inhibitors to analyse changes in conformation and phosphorylation that occur after Abl activation. I could show that the Abl SH2-kinase interface enabled trans-phosphorylation of the activation loop and disruption of the SH2âkinase interaction abolished its phosphorylation. To determine the sufficiency of targeting the SH2-kinase interface for Bcr-Abl inhibition, high-affinity monobodies targeting the kinase-binding surface of the Abl SH2 were developed in collaboration with the laboratory of Shohei Koide and their effects tested. The new monobodies inhibited Bcr-Abl kinase activity in vitro and in cells, and induced cell death in a CML cell line. We further used one of the monobodies to develop a screening assay based on the Homogeneous Time Resolved Fluorescence (HTRF) principle. We sucessfully showed that some specific donor/acceptor combinations could serve as a basis for high-throughput screening. Finally, we have studied allosteric regulation of several other cytoplasmic tyrosine kinases with an emphasis on the Btk kinase. We showed that the Btk kinase was strongly dependent on its SH2 domain for activation loop autophosphorylation, a feature that was also observed in transfected mammalian cells. The long-termgoal is to identify new allosteric regulations in oncogenic cytoplasmic tyrosine kinases that will help developing new types of therapeutics for hematological malignancies or solid tumors. |
