Abstract
In mice, the action of the DNA repair enzyme alkyladenine DNA glycosylase (AAG) has been found to cause cell death in different organs, including the retina, spleen, thymus, and cerebellum, in response to alkylation-induced DNA damage. Additionally, AAG has been associated with damage resulting from ischaemia/reperfusion (I/R) events in the liver, brain, and kidney. Consequently, there is a need to develop small molecule inhibitors of AAG to further investigate the biological mechanisms of cellular damage caused by AAG, as well as for potential drug leads for retinal degeneration, I/R-related tissue damage, or as protective agents for patients undergoing alkylative chemotherapy who display increased AAG activity. Such inhibitors could also serve the opposite effect by acting as an alkylating agent (TMZ) sensitiser in paediatric glioblastoma (GBM).
Although two DNA oligomers containing etheno-cytidine or an abasic pyrrolidine have been found to effectively inhibit AAG in vitro, they are not viable drug leads in vivo due to their size and charged nature, which makes them poorly permeable to membranes and susceptible to degradation by nucleases. However, these oligomers' motifs and the examination of the enzyme active site led to the conception of two small drug-like pyrrolidine-based inhibitor candidates, 2-(hydroxymethyl)pyrrolidines and 4-(hydroxymethyl)pyrrolidines, which could be developed as drug leads. A hit compound discovered by previous research in the group served as the starting point for further optimisation.
This study optimised the synthetic routes to these inhibitor candidates and developed a bioassay to evaluate their enzymatic activity, including the synthesis of abasic site binding probes and optimisation of a previously developed bound fluorophore assay. Although the synthetic route to the 2-(hydroxymethyl)pyrrolidines failed during the attachment of DNA base-mimicking aryl groups, three 4-(hydroxymethyl)pyrrolidines nucleoside mimetics were successfully synthesised, bearing bicyclic heteroaromatic groups to represent a DNA base. All inhibitor candidates were assessed for their bioactivities but showed no inhibitory activity, including the previously discovered hit compound when re-synthesised. Investigation into the cause of the hit compounds failure was also undertaken.
Overall, this study represents progress in developing AAG inhibitors and provides a basis for further research into the biological effects of AAG and its potential as a drug target for various diseases.