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Title: Characterisation of antagonist binding sites on chemokine receptor CCR4
Author: Viney, Jonathan Mark
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2013
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CCR4 is a chemokine receptor notably expressed on T helper 2 and regulatory T cells. CCR4 binds the chemokines CCL17 and CCL22. These are involved in T cell homeostasis and inflammatory diseases including asthma and atopic dermatitis, making CCR4 a potential therapeutic target. Previous studies suggested that CCL22 is dominant over CCL17 with respect to ligand-induced internalisation and desensitisation of CCR4. The biology of CCR4 was investigated in this project using point mutational studies. A C-terminal lysine within the conserved helix VIII region was determined to be dispensable for CCL22-induced chemotaxis but required for CCL17-induced chemotaxis, suggesting that the two chemokines stabilised distinct receptor conformations. The highly conserved GluVII:06 of helix VII was shown to be critical for chemokine binding and receptor function. Seven small molecule allosteric antagonists of CCR4, supplied by GlaxoSmithKline, were hypothesised to bind either a classical intrahelical site (site 1) within the receptor or a novel intracellular site (site 2). 22 amino acids were predicted to be involved in the binding of the antagonists. Antagonist binding was indirectly investigated by inhibiting either function or chemokine binding of the receptor mutants. Mutation of leucine 118 in transmembrane helix III significantly reduced CCR4 sensitivity to site 1 antagonism in chemotaxis and chemokine-binding assays. Mutants of phenylalanine 305 and leucine 307 at the end of transmembrane helix VII also showed a reduction in antagonist 2 sensitivity. Direct investigation of the effects of mutation on antagonist binding was performed using tritium-labelled antagonists. Mutation of GluVII:06 prevented site 1 antagonist binding to CCR4. Further investigation of site 1 antagonist binding was hindered by high non-specific binding of the compounds. A low-affinity site for the tritium-labelled site 2 antagonist was identified on untransfected cells, possibly within endogenously expressed chemokine receptors. This antagonist therefore may have potential as a broad-spectrum chemokine receptor inhibitor.
Supervisor: Pease, James ; Hall, David Sponsor: Biotechnology and Biological Sciences Research Council ; GlaxoSmithKline
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available