Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.555569
Title: Biological analysis of NRH quinone oxidoreductase 2 (NQO2) using novel inhibitors
Author: Caraher, Mary Clare
Awarding Body: University of Manchester
Current Institution: University of Manchester
Date of Award: 2012
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Abstract:
NQO2 is a member of the oxidoreductase family. It is a cytosolic, flavin-dependent, ubiquitous protein that is expressed in many human tumours. Liao and Williams-Ashman discovered the enzyme 51 years ago, however its cellular role has yet to be fully characterised. Intracellular NQO2 activity has been associated with various diseases; cancer, malaria and neurodegenerative disorders including schizophrenia, Alzheimer’s disease and Parkinson’s disease. Some of these associations have been related to the stability and activity of redox-sensitive factor NFκB, as well as tumour suppressor p53. Hence, suggesting NQO2 could be an important therapeutic target. However, the full extent of NQO2’s role in mediating these diseases has not been elucidated. In order to gain more insight into the cellular function of NQO2, a series of potent novel NQO2 inhibitors were identified from the NCI database. These compounds were assessed for their inhibition of NQO2 activity in cell-free systems as well as in cellular environments. Their inhibition potency of intracellular NQO2 was determined utilising NQO2’s ability to activate prodrug CB1954. These novel inhibitors were then used as a tool to evaluate NQO2’s role in the NFκB pathway; measuring TNFα-induced NFκB luciferase activity, using western blotting to assess NQO2’s impact on IκB-α and IKKβ protein stability, and the griess assay to analyse downstream product iNOS. In addition, regulation of redox status by NQO2 was determined using flow cytometry analysis. To help understand the underlying physical processes involved in binding interactions with chaperone proteins such as p53, NQO1 and NQO2 enzyme kinetics were studied and analysed using the Hill Equation as an indicator of cooperativity. Amongst the most potent inhibitors of intracellular NQO2 activity were NCI compounds NSC71795, NSC164016 and NSC305836 with IC50 values of 54nM, 2.3μM and 18μM compared to resveratrol, the ‘classic’ NQO2 inhibitor, with an IC50 value of 150μM. Each of these inhibitors attenuated TNFα-induced NFκB activity, as well as reduced intracellular iNOS concentration, and they partially protected IκB-α from proteasome degradation. NQO2’s involvement in some of these observations was confirmed through siRNA experiments. However, there was no clear modulation of IKKβ protein expression by NQO2. Inhibition experiments also identified NQO2 can alter the cellular redox. Additional findings include, NRH appears to modulate NFκB activity in an NQO2-dependent fashion, and the observed NQO2 regulation of NFκB activity and redox status appears to be more prevalent in tumour cells as opposed to normal cells. Kinetic analysis of NQO1 and NQO2 activity suggests the active sites in both enzymes are non-equivalent. The calculated Hill coefficients indicate the inhibitors induce negative cooperativity within the enzymes, which is altered in the additional presence of electron donor. Western blot analyses demonstrate inhibitor-induced destabilisation of p53 can be protected by electron donor. Thus, potential changes to the enzyme’s structure as a result of cooperativity could influence binding and stability of p53.In summary, the identified novel NQO2 inhibitors have helped to evaluate NQO2’s role in the NFκB pathway, and strengthen the theory this is achieved through alteration of cellular redox. Also, this study has re-opened the discussion that the active sites of NQO1, and NQO2, are non-equivalent as originally suggested by Ernster and colleagues.
Supervisor: Stratford, Ian. ; Nolan, Karen. Sponsor: BBSRC
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.555569  DOI: Not available
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