Pharmacological insights into C-­‐C motif chemokine receptor 5 mediated chemotaxis
Aim: Despite being well validated as a therapeutic target, no chemokine receptor antagonists to be used as therapeutic agents in inflammatory or metastatic disease have made it to market. This is in part due to receptor redundancy, but also a lack of understanding with regard to cytoplasmic signal transduction linking activated chemokine receptors to chemotaxis. Resolving signal transduction pathways in model chemokine receptor systems may allow intracellular drug targets to be identified, bypassing the difficulties associated with extracellular chemokine receptor blockade. Methodology: Experimentation was undertaken in THP-­‐1 monocytes expressing the chemokine receptors CCR5 and CCR1 and in stably CCR5-­‐transfected HeLa and CHO cell lines. Small molecule inhibition and protein overexpression was used before chemotaxis and calcium release assays to measure cellular responses. Immunocytology was used to determine the effect of protein blockade on receptor internalisation, protein localisation and the formation of cellular structures associated with migration. Experiments were also performed in activated primary tissue for comparative analysis and validation of results in normal human tissue. Results: A systematic blockade of signalling proteins by small molecule means revealed that Gβγ, ERK1/2, p38 and PI3K are not required for CCL3 stimulated monocyte migration. GRK2 and PKC inhibition along with internalisation blockade showed antagonistic effects on the ability of cells to migrate, suggesting arrestin dependent signalling was involved in chemotaxis. Inhibition of dynamin, Grb2 and non-­‐receptor tyrosine kinases were equally effective at blocking migration in THP-­‐1 cells but less effective at blocking CXCL11 stimulated migration in activated PBLs. Conclusions: This study has shown that CCL3 stimulated chemotaxis through CCR5 does not occur through typical G-­‐protein mediated signalling, but maybe therapeutically accessible by inhibition of dynamin and Grb2. Additionally the differences in dynamin inhibitor efficacy suggest that the production of migration specific dynamin inhibitors may be possible. Overall the research in this thesis has identified novel targets for therapeutic intervention in diseases where dysregulation of chemokine receptor mediated migration are causative.
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