TOMLINSON, CHARLES,WILLIAM,ERNEST (2021) Structural and Functional Relationships of Retinoic Acid Binding Proteins and Synthetic Retinoid Derivatives. Doctoral thesis, Durham University.
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Retinoic acid receptors (RARs) offer an unparalleled drug target for many underserved neurodegenerative and oncological pathologies due to their wide ranging, and potentially highly tuneable, influence over cell differentiation. This influence is based on the interaction of RAR and retinoid-X receptor (RXR) proteins in a heterodimer, which becomes an active transcription factor once retinoid ligands are bound, enabling binding at the retinoic acid response element (RARE) DNA sequence. Signalling output is influenced by the cellular retinoic acid binding proteins CRABPI and CRABPII, which mediate non-genomic signalling and perform nuclear import of retinoic acid, respectively. Current retinoid based treatments extend to minor modifications of all-trans retinoic acid, and a small number of synthetic derivatives with largely unknown modes of action. Recently developed synthetic retinoid derivatives, and their solvatochromic fluorescent counterparts, are the basis for the development of a tool kit for retinoid binding investigation.
A solvatochromic fluorescence assay for interpreting ligand affinity to retinoic acid binding proteins has been developed and validated, based on the activity of key synthetic retinoid DC271. The fluorescence displacement assay is backed up with rigorous structural evidence demonstrating ligand binding to CRABPII and CRABPI-L29C. The binding of synthetic retinoids to both CRAB and RAR proteins is characterised through use of the assay, and the resulting data reveals key chemical functionalities responsible for changes to binding affinities. Further work has aided in the validation and structural characterisation of a novel protein-protein interaction between CRABPII and cyclin D3, including key retinoic acid binding responses.
X-ray crystal structures of CRABPI-L29C with bound synthetic retinoid DC645 (2.4 Å), and with fatty acids MYR and TDA (1.6 Å) are reported. Co-crystal structures of nuclear transport protein CRABPII were determined with synthetic retinoids DC360 (1.8 Å), DC271 (1.5 Å), EC19 (2.8 Å), DC479 (1.8 Å) and DC645 (1.7 Å) in a variety of crystal forms. CRABPII mutant R29AK30A structural determination (1.35 Å) offers key insight into the folding of the protein, and its subsequent binding behaviour with retinoic acids in solution. These studies have significantly improved the understanding of interactions between synthetic retinoids and cognate binding proteins, and will inform the design of future target-specific candidate molecules.
|Item Type:||Thesis (Doctoral)|
|Award:||Doctor of Philosophy|
|Keywords:||Retinoids; Fluorescence; CRABPII; CRABPI; RAR; RXR|
|Faculty and Department:||Faculty of Science > Chemistry, Department of|
|Copyright:||Copyright of this thesis is held by the author|
|Deposited On:||06 May 2021 11:23|