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Title: Target identification in cell lysates and live cells using affinity approaches
Author: Negoita-Giras, Gabriel
ISNI:       0000 0004 2712 496X
Awarding Body: Institute of Cancer Research (University Of London)
Current Institution: Institute of Cancer Research
Date of Award: 2011
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As part of the BRAF inhibitor discovery programme at the lnstitute of Cancer Research, three pyrazine compounds were identified that exhibited good antiproliferative activities against the WM266.4 melanoma cell line, although they did not inhibit BRAF. As the potential targets of these compounds could be useful in the treatment of melanoma, a proteomic strategy was designed and implemented to purify and identify these proteins by affinity pulldown. Pulldown procedures from cell lysates and living cells were employed using the inhibitors immobilised with polymer and biotinylated or modified with click chemistry-and conjugated. Several affinity and control matrices, alkynyl and biotinylated probes were obtained by varying the type of linker, the linker length and the modified ligand. Twelve polymer-immobilised pyrazines and two corresponding inactive controls were synthesised, and these solid bound inhibitors were used in affinity pulldowns. These experiments led to the identification of ALDH1A3, VIME, PLEC, ENAH, VASP and several other proteins, that were purified as potential melanoma targets. These proteins were resolved by SDS-PAGE, identified in MS experiments, quantified using SILAC-MS and scored based on the SILAC q uantification experiments. For ALDH1A3, validation of the cellular phenotype (inhibition of proliferation), induced by the original pyrazine inhibitor, by siRNA gene knockdown was peformed. This experiment demonstrated the importance of ALDHlA3 in cellular proliferation of WM266.4 cells. A further examination of the inhibitortarget interaction using a competitive binding/S|LAC-MS approach suggested that EFHD2, CPOX and NME4 could be genuine target proteins of a different inhibitor. In an alternative approach several biotinylated conjugates and alkynyl probes of these compounds were synthesised, that can be used to pull down the target proteins from living cells. The immobilisation on either solid supported streptavidin or using click chemistry was assessed for these probes. These conjugates could prove to be important tools in order to assess the differences between pull-downs in the cell's native environment and in the cell lysates
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available