Usage
  • 10 views
  • 7 downloads

From Activation to Inhibition: p53 and PNKP as Targets for Cancer Therapy

  • Author / Creator
    Omar, Sara I
  • Cancer remains a challenging disease to tackle. The heterogeneity and evolution of the disease necessitates the development of diverse approaches to treat cancer. In this thesis, two different approaches have been undertaken to address this issue. The first aim was to activate the master tumor suppressor protein, p53. The second aim was to inhibit polynucleotide kinase phosphatase, a DNA repair enzyme.The first target, p53, has been coined the 'guardian of the genome'. It is a transcription factor that orchestrates protective actions in response to cellular stress. p53 pathways are always inactivated in cancer cells. This is not surprising given the highly significant role of the protein in cells. p53 is inactivated due to mutations in the TP53 gene in about 50% of cancers. This makes p53 the most mutated protein in all cancer types. Mutant p53 accumulates in cells. Reactivation of mutant p53 can, therefore, induce a massive response leading to cancer cell death. This effect can theoretically be very selective to abnormal cells carrying the mutant. Only a few small molecules that restore the wild-type activity to mutant p53 have been identified. One of these molecules, called APR-246, is currently in clinical trials. The exact effect of APR-246 on the structure of mutant p53 is not fully understood. Four aims arediscussed in this thesis: (1) understanding the interaction of known mutant p53 activators with the protein, (2) understanding the effect of three high frequency single-point mutations on the structure and DNA binding ability of the protein (3) understanding how the active form of APR-246 alters the DNA binding of mutant p53 and (4) virtually screening for novel mutant p53 activators.Atomistic models of three p53 mutants carrying the R175H, G245S or R273H mutation were created. The models revealed that the flexibility of loops L1, L2 and L3 in the DNA bindingdomain of p53 was correlated. Binding energy calculations also revealed that the mutations change the binding profile of the mutant proteins with DNA. Another striking similarity wasthat all three mutants exhibited distortions in their alignment with DNA compared to the wild-type protein. These distortions might further contribute to alterations in the transcriptional activity of p53 mutants.A previous study indicated that alkylating mutant p53 activators react with C124. We docked both alkylating and non-alkylating mutant p53 activators at this site. Docking results suggested that alkylating activators do not directly interact with C124 but their reactive moieties were directed towards the thiol of C124. However, non-alkylating activators were predicted to directly interact with C124. Poses of the non-alkylating ligands are logical given that they were predicted to interact with the proposed reactivation site. On the other hand, poses of the alkylating modifiers could be considered transitional state before reaction occurs. The covalently modified R175H and R273H p53 mutants were also simulated. It was observed that the wild-type and drugged mutants 'sat' on the DNA via a 'base'. However, there was a loss of interactions between regions of this base, in the mutant variants, and DNA. These results suggest that the alkylation of mutants' C124 anchors the L1 loop of p53 to DNA. Therefore, the modified proteins were aligned with DNA in a manner similar to wild-type p53, unlike the mutants.The second target, polynucleotide kinase phosphatase, is involved in repairing cellular DNA. Inhibition of this novel target promises to have synergistic effects in combination with current cancer treatment modalities that act by damaging DNA. In addition, this DNA repair enzyme has been found to be synthetically lethal to cells with phosphatase and tensin homolog (PTEN) or Src Homology region 2 domain-containing Phosphatase-1 (SHP-1) deficiency, which are common in some types of cancer. Virtual screening of a 3.7 million compoundswas performed in a multiple-technique approach. The first step of screening was based on pharmacophore modelling followed by pharmacophore-assisted docking. Six of the top hits were purchased for experimental validation. The measured dissociation constants from tryptophan fluorescence quenching assays ranged from 55 to 450 nM. Further, one of the compounds had an IC50 of ~13 μM. Iterations of structural optimization of these molecules could yield a potent polynucleotide kinase phosphatase inhibitor that could be used in combination cancer therapy.

  • Subjects / Keywords
  • Graduation date
    Spring 2019
  • Type of Item
    Thesis
  • Degree
    Doctor of Philosophy
  • DOI
    https://doi.org/10.7939/r3-nqyk-9r35
  • License
    Permission is hereby granted to the University of Alberta Libraries to reproduce single copies of this thesis and to lend or sell such copies for private, scholarly or scientific research purposes only. Where the thesis is converted to, or otherwise made available in digital form, the University of Alberta will advise potential users of the thesis of these terms. The author reserves all other publication and other rights in association with the copyright in the thesis and, except as herein before provided, neither the thesis nor any substantial portion thereof may be printed or otherwise reproduced in any material form whatsoever without the author's prior written permission.