Abstract
The base excision DNA repair (BER) enzyme alkyladenine glycosylase (AAG) can drive DNA damage-induced cell death in specific cell types in mice and can induce frameshift mutagenesis and microsatellite instability in yeast and in human cells. It was hypothesised that humans with overactive AAG, or who encounter higher levels of alkylating agents in the form of pollution, diet or chemotherapy, or suffer an ischaemic reperfusion event such as a stroke, may incur increased tissue damage through this mechanism. An inhibitor of AAG is required to further study this mechanism and form a potential lead for future drug discovery. In previous work, to discover an inhibitor, a published X-ray co-crystal structure of AAG was used in a virtual screen of two million compounds for potential binding activity. Of the top 49 virtual hits, one real hit triazole-thione-based inhibitor (UNIS00021) with an IC50 of ~60 µM was identified in a biochemical assay. In this thesis, efforts to design and synthesise analogues of UNIS00021 with improved potency against AAG are described. Successful divergent syntheses were developed which provided access to: 1. analogues varying at the alkyl group of the amide (six different amides); 2. analogues with a free amine in place of the amide and with variation of the length of the alkyl linkage group (five different amines); and 3. analogues bearing a C5-methyl group instead of thiol/thione at the core (one cyclohexylamide triazole). Work was also begun on the synthesis of analogues varying the N4-CH2-aryl group but was not completed due to time constraints. Two main types of microplate biochemical assay were investigated for assessment of the candidate inhibitors’ potencies against AAG using: 1. a surface-bound fluorescein-conjugated substrate DNA-oligomer; and 2. a free substrate oligomer and LCMS. Despite much experimentation, these assays continued to show inconsistent and irreproducible inhibition curves so it was not possible to make conclusions about the candidate inhibitors’ potencies.