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Structural analysis of LMO2 for the development of a small molecule inhibitor

Sewell, Helen (2010) Structural analysis of LMO2 for the development of a small molecule inhibitor. PhD thesis, University of Leeds.

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Sewell_H_LIMM_PhD_2010.pdf
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Abstract

LMO2 is a nuclear LIM-only protein encoded by a gene located on chromosome 11p13 and was originally discovered through its activation due to specific chromosomal translocations in patients with T-cell acute lymphoblastic leukaemia (T-ALL). The specific chromosomal translocations occur with either the T-cell δ receptor gene (14q11) or T-cell β receptor gene (7q35) and result in aberrant LMO2 expression in T-cells. Transgenic mouse models of LMO2 induced T cell neoplasias showed that enforced LMO2 expression caused accumulation of immature thymic T cells, followed by clonal T cell tumours with long latency. LMO2 is therefore a specific therapeutic target as not only is it associated with chromosomal translocations but is also expressed in approximately 50% of T-ALL. The aims of this project were to structurally determine LMO2 for structure based drug development of small molecules that will target LMO2 protein-protein interactions. LMO2 could not be purified alone as removal of the fusion tag resulted in severe precipitation of the free LMO2. Consequently, LMO2 was co-expressed with an antibody single domain termed VH#576, and purified to a high yield and purity. A final construct of LMO2, spanning residues 9 to 147, bound to VH#576 has been crystallised and the structure solved, to a medium resolution of 3.3Å, using phase information from single anomalous dispersion (SAD) data in combination with molecular replacement. Using a mammalian two-hybrid mutagenesis screen, key VH#576 binding residues have been identified. This data can be used, in combination with the crystal structure of VH#576/LMO2ΔN7ΔC11, to produce a Pharmacophore model for in silico screening and lead drug discovery. In addition to the crystallography approach, NMR was also investigated as a means to collect structural data on VH#576, in solution. A protocol has been developed to isotopically label and purify VH#576 along with unlabeled LMO2, in order to increase the stability of the antibody single domain for NMR data acquisition. Solving the structure of VH#576 by NMR requires further data collection. Advantageously, NMR solution structures represent more physiological environment and comparison of a VH#576 NMR structure and the crystal structure would enable the detection of any crystallisation artifacts.

Item Type: Thesis (PhD)
Academic Units: The University of Leeds > Faculty of Medicine and Health (Leeds) > Institute of Molecular Medicine (LIMM) (Leeds)
Depositing User: Ethos Import
Date Deposited: 22 Sep 2010 09:28
Last Modified: 08 Aug 2013 08:45
URI: http://etheses.whiterose.ac.uk/id/eprint/1027

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