White Rose University Consortium logo
University of Leeds logo University of Sheffield logo York University logo

Characterization of the human Pif1 helicase as a potential cancer-therapy target

Dehghani-Tafti, Saba (2018) Characterization of the human Pif1 helicase as a potential cancer-therapy target. PhD thesis, University of Sheffield.

[img] Text (Thesis)
Thesis S Dehghani-Tafti.pdf
Restricted until 31 December 2024.

Request a copy
Text (embargo approval)
Available under License Creative Commons Attribution-Noncommercial-No Derivative Works 2.0 UK: England & Wales.

Download (237Kb) | Preview


Pif1 is a multifunctional 5´ → 3´ superfamily 1 (SF1) helicase that is conserved in eukaryotes and also some prokaryotes. In addition to unwinding activity, the protein is a G-quadruplex (G4) DNA binding protein and strand annealing enzyme. Eukaryotic Pif1 proteins have roles in mitochondrial and nuclear genome stability and S-phase completion that are best characterised in S. cerevisiae. Pif1 knock-out mice are normal and recent studies show that the functions of human Pif1 (hPif1) only become critical for survival of some tumour cells during oncogene-induced replication stress, indicating that the enzyme is a potential cancer therapy target. However, hPif1 is poorly characterised at the structural and biochemical level and its function(s) critical for tumour cell survival are unknown. Here, a structure-function study and a discovery campaign to identify small molecule inhibitors of hPif1 was initiated. A recombinant E. coli expression system was developed for hPif1 yielding milligrams to tens-of-milligrams of highly purified intact enzyme or truncation mutants encompassing helicase core (helicase domain, hPif1-HD). hPif1 crystal structures for the apo (1.4Å) as well as ATP hydrolysis ground (1.1Å) and transition state (3.9Å), representing structural events along the chemical reaction coordinate, were obtained. Comparisons with the structures of yeast and bacterial Pif1 reveal a conserved ssDNA binding channel in hPif1 that was demonstrated to be critical for single-stranded DNA binding during unwinding, but not the binding of G quadruplex DNA. Mutational analysis suggests that while the ssDNA-binding channel is important for helicase activity, it is not used in DNA annealing. Structural differences, in particular in the DNA strand separation wedge region, highlight significant evolutionary divergence of the human Pif1 protein from bacterial and yeast orthologues. Libraries of drug-like molecules and chemical fragments were screened in vitro for inhibitors of hPif1 helicase activity and, using a protein thermal shift assay, direct binding. This screening identified chemical entities, some of which demonstrated preferential inhibition of hPif1 helicase activity compared to the SF1 helicase hUPF1, with the potential to progress in development to effective inhibitors. The established hPif1 crystallisation conditions were used to exploit the XChem fragment-based screening service at the Diamond Light Source national facility. A 233.3 Da chemical entity, EN300-02473, was identified in a 1.73Å crystal structure bound in a pocket between domains 2A and 2B of hPif1, close to the ssDNA binding site. EN300-02473 and two structurally related analogues inhibited hPif1 helicase activity in vitro with IC50 values from 80-110 μM and significant selectivity relative to hUPF1. Together, the findings reported here will serve as a platform to dissect and target hPif1 functions in intact cells.

Item Type: Thesis (PhD)
Academic Units: The University of Sheffield > Faculty of Medicine, Dentistry and Health (Sheffield) > Medicine (Sheffield)
Depositing User: Miss Saba Dehghani-Tafti
Date Deposited: 25 Feb 2019 10:43
Last Modified: 25 Feb 2019 10:43
URI: http://etheses.whiterose.ac.uk/id/eprint/22834

Some parts of this thesis can be downloaded immediately using the 'Download' link(s) above. Other parts can be requested by using the 'Request a copy' link(s) above.
You can contact us about this thesis. If you need to make a general enquiry, please see the Contact us page.

Actions (repository staff only: login required)