Identification and characterisation of novel inhibitors of Polynucleotide Kinase 3'-Phosphatase

Polynucleotide kinase 3'-phosphatase (PnKP) is a DNA damage repair enzyme. It has two specific activities these being a 3’ DNA phosphatase and 5’ DNA Kinase. It is an integral part of DNA repair pathways including single strand break repair (SSB), base excision repair (BER) and non-homogenous end joining (NHEJ). The activity of PnKP restores broken DNA end termini to a form which allows ligation to proceed. A small molecule inhibitor of PnKP in specific cancer backgrounds such as cells with malfunctioning homologous recombination should be synthetically lethal. Alternatively, the same small molecule inhibitor could be dosed with radiotherapy to increase effectiveness of this treatment. Given the limited role of PnKP within the body, side effects should also be limited. In this thesis to identify a small molecule inhibitor of PnKP, three different compound collections (a fragment and the output of two different virtual screens) were tested in a newly developed 96 well plate format biochemical assay, utilising recombinantly expressed PnKP. This assay measures the production of phosphate produced from the cleavage of a specific 3’ DNA substrate. Parameters for the assay such as Km, Z’ and IC50 of key inhibitors were established and the assay was suitable for compound screening. A number of primary hits from the screening were identified with IC50 values ranging from 6 µM to 1.2 mM. Using a series of counter screening assays, false positives and non-specific inhibitors were removed from the cascade. Further biophysical characterisation of the hits was undertaken using microscale thermophoresis and waterlogsy NMR. KD values were measured which were similar to that of the IC50 obtained with the biochemical data. Crystal soaking was carried out and a novel 2.2 Å X-ray structure was obtained with one of the fragment hit compounds bound (UOS-00021277).