The ALK tyrosine kinase is expressed, as NMP/ALK fusion protein, in approximately 60% of Anaplastic Large Cell Lymphoma (ALCL) cases and in other cancers such as Non Small Cell Lung Cancer (NSCLC) and neuroblastoma. ALK kinase domain constitutive activation is responsible for the malignant transformation through stimulation of downstream survival and proliferation signalling pathways. A number of ALK inhibitors are under investigation, with crizotinib being recently approved in NSCLC. In particular, acquired resistance to kinase inhibitors is a serious problem in long-term cancer treatment including ALK+ cancers. Structural characterization of the ALK kinase domain and information about drug-protein interactions could be very useful in the rational design of new specific drugs for treatment of the ALK+ ALCL and other ALK+ tumours. This work has been focused on the production and purification of different forms of the ALK kinase domain to obtain good candidates for ALK structural studies by X-ray crystallography and STD-NMR. r-ALK proteins were expressed in Sf9 (Spodoptera frugiperda) insect cells using two different Baculovirus expression systems (Bac-to Bac and BacPAK expression systems). After optimization of the purification procedures, r-ALK proteins (WT and mutant forms) have been characterized obtaining three forms suitable for structural studies. In particular, P e NON-P forms of two purified r-proteins were used to screen about 20000 conditions for crystallization. STD-NMR results of the ALK 6 demonstrated the interaction between the ALK kinase domain and a new kinase inhibitor (R458). In addition, the r-ALK proteins has been used for screening of potential ALK inhibitors (R500B and R458), whose activity has been validated in ELISA kinase assay. In conclusion, the results of this work could pave the way to the development of new specific drugs for specific treatment of ALK+ diseases.
(2011). Structural studies on the anaplastic lymphoma kinase. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2011).
GAMBACORTI PASSERINI, CARLO
|Data di pubblicazione:||16-dic-2011|
|Co-tutore:||TARTARI, CARMEN JULIA|
|Titolo:||Structural studies on the anaplastic lymphoma kinase|
|Settore Scientifico Disciplinare:||MED/09 - MEDICINA INTERNA|
|Scuola di dottorato:||Scuola di Dottorato in Scienze Mediche Sperimentali e Cliniche|
|Corso di dottorato:||EMATOLOGIA SPERIMENTALE - 05R|
|Citazione:||(2011). Structural studies on the anaplastic lymphoma kinase. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2011).|
|Parole Chiave:||ALK, kinase, inhibitors, X-ray crystallography, STD-NMR|
|Appare nelle tipologie:||07 - Tesi di dottorato Bicocca post 2009|