Synthesis and structures of fluorescent retinoid receptor modulators

Abstract

Retinoids are endogenous molecules derived from Vitamin A that play key roles in a plethora of cellular processes throughout the human body, including differentiation and homeostasis, by mediating gene regulation in the nucleus. This process occurs when retinoids bind to the retinoic acid receptors (RAR) or retinoid X receptors (RXR), and the resultant complexes act as stable platforms for DNA transcription. However, the cascade of biological processes that is triggered by this binding event is still poorly understood, and new tools are required to understand this complex signalling pathway.
Endogenous retinoids are inherently fluorescent due to their conjugated polyene structure, but this fluorescence is typically too weak to use in cellular imaging or other biophysical studies. This thesis describes the synthesis of a library of highly fluorescent synthetic retinoids using a variety of synthetic methodologies, and a structure-based design approach. These fluorescent retinoids can be uniquely imaged in a cellular environment, and biological characterisation indicated that one of these retinoids, DC271, was able to enter the nucleus - the location of the RARs and RXRs.
The binding characteristics of the fluorescent retinoids was predicted, in silico, by docking each structure into the binding sites of the RARs and cellular retinoic acid binding protein II (CRABPII). This indicated that the fluorescent retinoids mimicked the binding poses of their endogenous forebears, and the docking solutions were used to predict the relative order of binding affinity for these proteins. These predictions were validated by the solution of the crystal structure of a fluorescent retinoid (DC360) bound to CRABPII, and the development of an in vitro fluorometric binding assay that utilised the fluorescence of the compounds to determine their binding affinity for CRABPII. The unique properties of these fluorescent retinoids can now be used to shed new light on the fascinating retinoid signalling pathway.