Application of an affinity chromatography toolbox to drug repurposing for cancer therapeutics
Cruickshank, Faye Louise
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Phenotypic screening of drug molecules relies on the generation of a specific response; however the means by which this is elicited often remains unknown. Affinity chromatography is a valuable tool in the discovery of drug binding partners and may even allow the elucidation of the wider interactome of the initial drug target. The introduction of easily cleavable linkers and affinity-independent elution protocols to affinity chromatography is of current interest, since they render the technique much more adaptable with respect to the characterisation of biologically active species of interest. This thesis details the application of a novel azobenzene linker developed by the Hulme group for use in affinity-independent chromatography. The first chapter reviews recent developments in affinity chromatography and describes the synthesis of an affinity linker toolbox with both affinity-dependent and affinity-independent linkers. These linkers are functionalised with an azide moiety for use in CuAAC coupling to alkynyl derivatives of bioactive small molecules and have been modified to include photoreactive groups giving a series of linkers for use in the identification of less abundant, or low affinity, proteins. The first drug investigated, anisomycin (ANS), is a small molecule which was initially introduced as an antibiotic drug (Flagecidin). At nanomolar concentrations ANS has been shown to affect the mitogen activated protein kinase (MAPK) pathways; downstream effects of these pathways are thought to play a role in a range of pathological disorders such as Alzheimer’s disease, cancer and spinal muscular atrophy (SMA). ANS is thus a candidate for drug repurposing. Although the downstream effects of MAPK/SAPK pathway activation induced by anisomycin are well-documented, the cellular target has yet to be revealed. Previous work by the Hulme group has shown that the N-propargyl anisomycin derivative (I) retains the biological activity of the lead compound ANS. Thus to evaluate the cellular protein targets, N-propargyl ANS (I) was coupled onto the linker toolbox to create an ANS affinity probe library as described in chapter 2. The second drug investigated, fingolimod, was introduced as an immunomodulating drug (Glienya) for the treatment of multiple sclerosis (MS). This small molecule has also been shown to have anti-cancer properties in a range of cancer cell lines; however the precise mechanism by which this is effected is unknown. Literature precedent shows that terminal modification of fingolimod generates analogues which still retain biological activity. Thus a novel fingolimod alkyne derivative (II) was synthesised and used to create an affinity probe library as described in chapter 3. Chapter 4 describes affinity pull-down experiments conducted with the aim of finding the protein target(s) of ANS and fingolimod, using the affinity probe libraries generated in chapters 2 and 3. This chapter concludes with a discussion of the implications of these findings and directions for future study.