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Pharmacological Activation of Glucose-6- Phosphate Dehydrogenase (G6PD) to Extend

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Pharmacological Activation of Glucose-6- Phosphate Dehydrogenase (G6PD) to Extend Pharmacological Activation of Glucose-6- Phosphate Dehydrogenase (G6PD) to Extend Lifespan Ashley Sue Ann Wong Thesis submitted in fulfilment of the requirements for the degree of Doctor of Philosophy School of Medical Sciences, Faculty of Medicine University of New South Wales Sydney Supervisors: Dr. Lindsay E. Wu University of New South Wales Sydney, NSW, Australia Professor David A. Sinclair University of New South Wales Sydney, NSW, Australia Harvard Medical School, Boston MA, United States of America March 2019 THE UNIVERSITY OF NEW SOUTH WALES SYDNEY Thesis/Dissertation Sheet Surname/Family Name: Wong First Name: Ashley Middle Name: Sue Ann Abbreviation for degree as given in the University calendar: PhD Faculty: Medicine School: Medical Sciences Thesis Title: Pharmacological Activation of Endogenous Glucose-6-Phosphate Dehydrogenase (G6PD) to Extend Lifespan __________________________________________________________________________________________________________ Aged cells face multiple biochemical challenges including diminished de novo nucleotide synthesis for faithful DNA replication, impaired redox capacity, and decreased NAD+ required to maintain sirtuin activity. A common thread running through these challenges is the requirement for the pentose phosphate pathway, which declines with age due to reduced activity of the rate-limiting enzyme glucose-6-phosphate dehydrogenase (G6PD). Over-expression of G6PD extends lifespan in Drosophila and mice, alongside improvements in metabolic homeostasis and motor coordination in old age. Outside of this genetic approach, pharmacological approaches to enhance endogenous G6PD activity could offer a clinically relevant opportunity to recapitulate the improved late-life health and extended lifespan observed during G6PD over-expression. We have performed a small molecule screen and identified a series of novel G6PD activators with drug-like properties that enhance G6PD activity, which work through allosteric modulation and stabilisation of the enzyme. These new compounds robustly increase lifespan in Caenorhabditis elegans, across four different structurally unrelated G6PD activators. These new compounds demonstrate for the first time that G6PD can be modulated by pharmacological approaches and represent a new class of drugs that can delay biological ageing and extend lifespan. Declaration relating to disposition of project thesis/dissertation I hereby grant to the University of New South Wales or its agents the right to archive and to make available my thesis or dissertation in whole or in part in the University libraries in all forms of media, now or here after known, subject to the provisions of the Copyright Act 1968. I retain all property rights, such as patent rights. I also retain the right to use in future works (such as articles or books) all or part of this thesis or dissertation. I also authorise University Microfilms to use the 350 word abstract of my thesis in Dissertation Abstracts International (this is applicable to doctoral theses only). ……………………………………. ……………………………………. Signature Witness Signature Date ……................ The University recognises that there may be exceptional circumstances requiring restrictions on copying or conditions on use. Requests for restriction for a period of up to 2 years must be made in writing. Requests for a longer period of restriction may be considered in exceptional circumstances and require the approval of the Dean of Graduate Research. FOR OFFICE USE ONLY Date of completion of requirements for Award: 1 COPYRIGHT STATEMENT ‘I hereby grant the University of New South Wales or its agents the right to archive and to make available my thesis or dissertation in whole or part in the University libraries in all forms of media, now or here after known, subject to the provisions of the Copyright Act 1968. I retain all proprietary rights, such as patent rights. I also retain the right to use in future works (such as articles or books) all or part of this thesis or dissertation. I also authorise University Microfilms to use the 350 word abstract of my thesis in Dissertation Abstract International (this is applicable to doctoral theses only). I have either used no substantial portions of copyright material in my thesis or I have obtained permission to use copyright material; where permission has not been granted I have applied/will apply for a partial restriction of the digital copy of my thesis or dissertation.' Signed ……………………………………………....................... Date ……………………………… AUTHENTICITY STATEMENT ‘I certify that the Library deposit digital copy is a direct equivalent of the final officially approved version of my thesis. No emendation of content has occurred and if there are any minor variations in formatting, they are the result of the conversion to digital format.’ Signed ……………………………………………....................... Date ……………………………… 2 ORIGINALITY STATEMENT ‘I hereby declare that this submission is my own work and to the best of my knowledge it contains no materials previously published or written by another person, or substantial proportions of material which have been accepted for the award of any other degree or diploma at UNSW or any other educational institution, except where due acknowledgement is made in the thesis. Any contribution made to the research by others, with whom I have worked at UNSW or elsewhere, is explicitly acknowledged in the thesis. I also declare that the intellectual content of this thesis is the product of my own work, except to the extent that assistance from others in the project's design and conception or in style, presentation and linguistic expression is acknowledged.’ Signed ……………………………………………....................... Date ……………………………… 3 Inclusion of publications statement UNSW is supportive of candidates publishing their research results during their candidature as detailed in the UNSW Thesis Examination Procedure. Publications can be used in their thesis in lieu of a Chapter if: • The student contributed greater than 50% of the content in the publication and is the “primary author”, ie. the student was responsible primarily for the planning, execution and preparation of the work for publication • The student has approval to include the publication in their thesis in lieu of a Chapter from their supervisor and Postgraduate Coordinator. • The publication is not subject to any obligations or contractual agreements with a third party that would constrain its inclusion in the thesis Please indicate whether this thesis contains published material or not. This thesis contains no publications, either published or submitted for publication ☒ Some of the work described in this thesis has been published and it has been documented ☐ in the relevant Chapters with acknowledgement This thesis has publications (either published or submitted for publication) incorporated ☐ into it in lieu of a chapter and the details are presented below CANDIDATE’S DECLARATION I declare that: • I have complied with the Thesis Examination Procedure • where I have used a publication in lieu of a Chapter, the listed publication(s) below meet(s) the requirements to be included in the thesis. Name Signature Date 4 Table of Content Thesis abstract .............................................................................. 9 Acknowledgements .................................................................... 10 Publications and Presentations ................................................. 11 List of Figures and Tables ......................................................... 13 Abbreviations ............................................................................. 17 Chapter 1 Introduction and Background ............................ 19 1.1 The Pentose Phosphate Pathway................................................. 20 1.1.1 Roles and function ........................................................................................ 20 1.1.2 Sentinel for defence against oxidative stress: NADPH ................................ 22 1.1.3 Cell growth, proliferation and DNA repair: Ribose-5-phosphate ................. 26 1.2 Glucose-6-Phosphate Dehydrogenase Enzyme .......................... 28 1.2.1 The G6PD gene/protein structure ................................................................. 29 1.2.2 Positive regulation of G6PD Enzyme Activity ............................................. 31 1.2.3 Negative regulation of G6PD Enzyme ......................................................... 33 1.3 Glucose-6-phosphate Dehydrogenase Deficiency ...................... 34 1.3.1 Epidemiology, Variants and WHO classification ......................................... 35 1.3.2 Phenotype and clinical manifestations .......................................................... 36 1.3.3 Management of G6PD deficiency ................................................................. 40 1.4 The Pentose Phosphate Pathway in Ageing and pathophysiology ……………………………………………………………………42 1.4.1 Diabetic phenotype and nephropathy ........................................................... 43 1.4.2 G6PD expression in cancer ........................................................................... 45 1.4.3 Neurodegenerative diseases .......................................................................... 47 1.4.4 Cardiomyopathies and cardiovascular diseases ............................................ 50 1.4.5 Ageing: healthspan and lifespan ................................................................... 51 5 1.5 Conventional therapies targeting the Pentose
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