Application of an affinity chromatography toolbox to drug repurposing for cancer therapeutics
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Date
28/06/2016Author
Cruickshank, Faye Louise
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Abstract
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.