Structural and Inhibitor Binding Studies of Tyrosyl-DNA Phosphodiesterase 1

Abstract

The work described in this thesis has focused on human tyrosyl-DNA phosphodiesterase 1 (TDP1), which is a DNA repair enzyme that is present in eukaryotes including humans. TDP1 is a monomeric protein from the phospholipase D (PLD) superfamily. TDP1 is involved in the repair of topoisomerase-mediated DNA damage during DNA replication and transcription. TDP1 is current inhibition target for improving the efficacy of cancer chemotherapy, as TDP1 also repair DNA damage caused by anticancer chemical agents in cancer cells. In addition to being a chemosensitizer target, a histidine to arginine (H493R) mutation in human TDP1 has also been shown to cause a neurodegenerative disease called spinocerebellar ataxia with axonal neuropathy (SCAN1). There are current efforts to develop activators as well as inhibitors of H493R TDP1 to treat the SCAN1 disease. Chapter 2 described the work in the production and purification of truncated and mutant human TDP1. Expression trials were performed by using the Escherichia coli expression system. Expression conditions including the concentration of the inducer isopropyl β-D-1- thiogalactopyranoside (IPTG) and incubation temperature were tested. Pure truncated TDP1149- 608 can be obtained by using affinity chromatography and followed by size exclusion chromatography. The production of H263L TDP1 was more challenging. No protein expression was observed when the expression trial was conducted with E. coli BL21 (DE3). Although soluble proteins were observed with E. coli ArcticExpress (DE3), the protein was found to be highly unstable despite the presence of protease inhibitors. In contrast, the production and purification of H493R TDP1 was more straightforward. Highly purified proteins could be achieved by using a similar procedure as for the truncated protein. Chapter 3 described the work in the development, optimisation and application of fluorescencebased binding assays to study TDP1-ligand binding interactions. Two fluorescence-based binding assays were investigated. These include thermal shift assay and intrinsic protein fluorescence spectroscopy. The results obtained by thermal shift assay were found to be highly variable. It was therefore decided that the thermal shift assay may not be suitable to study ligand binding TDP1. In contrast, as human TDP1 contains four tryptophan residues around 4- 8 Å distance from the active site, intrinsic protein fluorescence spectroscopy was successfully applied to study ligand binding to the protein. The ability of the assay to screen and quantify ligand binding was demonstrated by a series of new TDP1 inhibitors. The drawbacks of the assay were also demonstrated. The use of the intrinsic protein fluorescence assay to study ligand binding to H493R TDP1 was also studied. By comparing the results obtained with wildtype and mutant TDP1, it showed that some TDP1 inhibitors may bind to the active site of the enzyme, and some may be binding elsewhere in the substrate binding pocket. Finally, the use of small nucleophiles as potential activators of H493R TDP1 was also investigated although the results were negative. Chapter 4 described the work in the use of X-ray crystallography to obtain crystal structures of TDP1. Crystal structure of human TDP1 was successfully obtained. The structure was found to be the same as the published structure of human TDP1. However, efforts to obtain crystal structure of TDP1 in the presence of its inhibitor were found to be unsuccessful. Although both the ligand soaking and co-crystallisation methods were attempted, it was unable to obtain any structures of TDP1 in the presence of its inhibitor. Overall, the work described in this thesis has furthered the progress in the quest to develop inhibitors of TDP1 as a sensitizer for chemotherapy drugs. The optimised protocols in the production and purification of human TDP1, the binding assays and the protocols to obtain human TDP1 crystal structures will enable medicinal chemists to obtain structural-activity relationships of TDP1 inhibitors in the future.