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.

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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

1.5 Conventional therapies targeting the Pentose phosphate pathway

……………………………………………………………………53

1.5.1 G6PD inhibiting agents ...... 53

1.5.2 G6PD activating agents ...... 56 Chapter 2 High-throughput screen for small molecule G6PD activators ...... 58

2.1 Introduction ...... 59

2.2 Materials ...... 61

2.2.1 Materials and reagents ...... 61

2.2.2 Instruments...... 62

2.3 Experimental Methods ...... 63

2.3.1 G6PD kinetic studies ...... 63

2.3.2 HTS screen stop solution optimization ...... 63

2.3.3 G6PD enzyme titration for HTS screen ...... 64

2.3.4 G6PD enzyme DMSO tolerance test ...... 64

2.3.5 HTS G6PD enzyme activator screen ...... 65

2.3.6 G6PD enzyme activator/inhibitor HTS hits validation screen ...... 66

2.3.7 G6PD activator/inhibitor Hits EC50/IC50 determination ...... 67

2.4 Results ...... 68

2.4.1 G6PD enzyme assay optimization for HTS ...... 68

2.4.2 G6PD HTS validation ...... 79

2.4.3 G6PD HTS for enzyme activators ...... 88

2.5 Discussion ...... 92

Chapter 3 Characterising ASW Compounds ...... 103

3.1 Introduction ...... 104

3.2 Materials ...... 105

3.2.1 Materials and reagents ...... 105

3.2.2 Instruments...... 106

3.3 Experimental Methods ...... 107

3.3.1 G6PD enzyme activity assay ...... 107

3.3.2 G6PD kinetic studies ...... 107

3.3.3 Construction, design and site-directed mutagenesis of recombinant G6PD …………………………………………………………………………….108

3.3.4 Bacterial transformation procedure ...... 109

3.3.5 Computational molecular docking ...... 110

3.3.6 Expression and purification of G6PD enzymes ...... 111

3.3.7 SDS-PAGE and Western blot analysis ...... 111

3.4 Results ...... 112

3.4.1 ASW compounds stabilise, rather than activate G6PD enzyme activity …………………………………………………………………………….112

3.4.2 Lead G6PD-stabilising ASW compounds predicted to affect enzyme dimerization resulting in altered G6PD enzyme kinetics ...... 119

3.4.3 G6PD-stabilising ASW compounds exhibit G6PD variant specificity for action …………………………………………………………………………….127

3.5 Discussion ...... 134 Chapter 4 Effect of novel G6PD-stabilisers on health and lifespan ...... 143

4.1 Introduction ...... 144

4.2 Materials ...... 146

4.2.1 Materials and reagents ...... 146

4.3 Experimental Methods ...... 148

4.3.1 Nematode Growth Media (NGM) preparation ...... 148

4.3.2 Nematode Synchronization ...... 148

4.3.3 Caenorhabditis elegans survival assay ...... 149

4.3.4 Antioxidant capacity assessment ...... 149

4.3.5 G6PD enzyme activity assay ...... 150

4.3.6 Caenorhabditis elegans pharyngeal and thrashing rate assays ...... 150

4.3.7 Caenorhabditis elegans stress resistance assay ...... 151

4.4 Results ...... 152

4.4.1 Lifespan of C. elegans is improved with ASW compound treatment ...... 152

4.4.2 Improved lifespan observed in ASW compound-treated nematodes is not paired with an improvement in physical activity or healthspan ...... 168

4.4.3 ASW compounds extends lifespan in a G6PD-dependent manner, but do not rescue age-associated decline of G6PD activity ...... 173

4.5 Discussion ...... 183

Chapter 5 General discussion ...... 195

5.1 Study significance...... 196

5.2 Evaluation of experimental direction ...... 197

5.2.1 High-throughput small molecule screen ...... 198

5.2.2 ASW drug G6PD enzyme activity modulation...... 199

5.2.3 Choice of animal model ...... 202

5.3 Clinical relevance of G6PD stabilisers ...... 204

5.4 Future directions ...... 207

5.5 Conclusion ...... 210

Supplementary figures ...... 211

References ...... 230

Thesis abstract

Acknowledgements

I would like to thank my supervisors Dr. Lindsay Wu and Professor David Sinclair for their guidance, incredible support and patience throughout my PhD journey. Dr. Wu always made sure I was on track with my project whilst allowing me freedom to mould my project the way I see fit and encouraging me to do my best, guiding me through research techniques, experimental planning, scientific writing and much more. Professor Sinclair encouraged me to broaden my knowledge and inspired me to improve my research skills, always being supportive and giving me great advice on both my project and for my career. Furthermore, both my supervisors were my source of inspiration, encouraging me to strive to better myself, and to soldier on through the ups and downs of my PhD. They were supportive and encouraged me to not only focus my entire time on my project, but to actively learn and travel to broaden my knowledge and experience, which I am incredibly grateful for.

My gratitude also goes to my lab colleagues past and present: Abhi, Jin, Hassina, Catherine, Lynn, Jon, Tim and many others, for their support, help, advice and for making the lab environment pleasant and fun to be around. They have seen me at my best and worst during the course of our time together, and still gave me their friendship, and I feel lucky to have had them with me during this journey. I would also like to extend my gratitude to everyone in Wallace Wurth 3 East UNSW for the friendships made, support given and the overall pleasant atmosphere throughout my PhD years.

Finally, I would like to thank my parents Terry and Lily for their love, guidance and advice over the years, and for giving me the freedom to choose my own path and fully supporting my decisions. Thank you William, Ian and Charmaine my siblings, my dog Simba, and all my friends for your patience and understanding when I prioritised my work. I would also like to thank my partner Vincent for your patience and support. You are a legend.

Publications and Presentations

Cagnone, Gael L. M., Tsai, Te-Sha, Makanji, Yogeshwar, Matthews, Pamela, Gould, Jodee, Bonkowski, Michael S., Elgass, Kirstin D., Wong, A. S. A., Wu, L. E., McKenzie, M., Sinclair, D. A. and John, J. C. S. (2016). Restoration of normal embryogenesis by mitochondrial supplementation in pig oocytes exhibiting mitochondrial DNA deficiency. Scientific Reports 6: 23229.

Wong, A. S. A., Wu, L. E. and Sinclair, D. A. (2016). Small molecule activation of the pentose phosphate shunt for Pharmacological extensions in lifespan. Oral presentation at the Biology Domain Symposium On Metabolism, Charles Perkins Centre, Sydney, Australia.

Wong, A. S. A., Wu, L. E. and Sinclair, D. A. (2017). Pharmacological activation of endogenous Glucose-6-phosphate dehydrogenase (G6PD) with novel drugs to extend lifespan. Poster presentation at the Australian Biology of Ageing Conference, Sydney, Australia.

Wong, A. S. A., Wu, L. E. and Sinclair, D. A. (2017). Pharmacological activation of endogenous Glucose-6-phosphate dehydrogenase (G6PD) with novel drugs to extend lifespan. Poster presentation at the 2nd Interventions in Ageing, Understanding Mechanisms & Compressing Morbidity in Aging Humans, Cancun, Mexico.

Wong, A. S. A., Wu, L. E. and Sinclair, D. A. (2018). Pharmacological activation of endogenous Glucose-6-phosphate dehydrogenase (G6PD) with novel drugs to extend lifespan. Poster presentation at 2018 IUBMB Seoul, 24th IUBMB Congress & 15th FAOBMB Congress, Integrating Science for Bio-Health Innovation, Seoul, South Korea.

Wong, A. S. A., Wu, L. E. and Sinclair, D. A. (2018). Pharmacological activation of endogenous Glucose-6-phosphate dehydrogenase (G6PD) with novel drugs to extend lifespan. Oral presentation at the Australian Biology of Ageing Conference, University of Queensland, Brisbane, Australia.

Wong, A. S. A., Wu, L. E. and Sinclair, D. A. (2018). Pharmacological activation of endogenous Glucose-6-phosphate dehydrogenase (G6PD) with novel drugs to extend lifespan. Poster presentation at the Cell Symposia: Aging and Metabolism, Sitges, Spain.

Wong, A. S. A., Wu, L. E. and Sinclair, D. A. (2018). Pharmacological activation of endogenous Glucose-6-phosphate dehydrogenase (G6PD) with novel drugs to extend lifespan. Poster presentation at the CSHL Meetings: Mechanisms of Aging, Cold Spring Harbour Laboratory, New York, USA.

Wong, A. S. A., Wu, L. E. and Sinclair, D. A. (2019). Pharmacological activation of endogenous Glucose-6-phosphate dehydrogenase (G6PD) with novel drugs to extend lifespan. Poster presentation at the Australian Biology of Ageing Conference, University of Sydney, Australia.

List of Figures and Tables

Figures Page 1.1.1.1 Summary of the Pentose Phosphate Pathway 21 The G6PD enzyme sequence is highly conserved in a variety of 1.2.1 28 species. Conserved regions in amino acid alignment of G6PD homologues in 1.2.1.1 29 humans, mouse, fruit fly, bacteria and nematodes. Optimization of G6PD concentration for high-throughput assay 2.4.1.1 69 development.

Km determination for G6PD substrates for high-throughput 2.4.1.2 72 screening optimization. Optimization of GdnHCl stop solution concentration for G6PD HTS 2.4.1.3 76 development. 2.4.1.4 G6PD enzyme DMSO tolerance test for HTS optimization. 78 2.4.2.1 Plate layout for the G6PD HTS. 80 G6PD HTS optimization for enzyme pretreatment and reaction 2.4.2.2 82 runtime. 2.4.2.3 High-throughput G6PD activator screen parameters. 86 Effect of ASW compounds on G6PD enzyme activity with varied 3.4.1.1 113 lengths of enzyme pretreatment Recombinant human wild-type G6PD loses activity over time in 116 3.4.1.2 vitro.

Lead G6PD activators function as enzyme stabilisers preventing loss 118 3.4.1.3 of enzyme activity over time.

Ligand-enzyme 2D interaction map with G6PD enzyme stabilising 3.4.2.1 122 ASW compounds. Effect of G6PD-stabilising ASW small molecules on G6PD enzyme 3.4.2.2 126 kinetics. 3.4.3.1 G6PD mutant variant plasmid generation. 128 SDS-PAGE analysis of purified G6PD deficiency variants with 3.4.3.2 130 measurement of G6PD enzyme activity. 13

3.4.3.3 G6PD-stabilising small molecules exhibits G6PD variant specificity. 132 4.1 The G6PD enzyme sequence is highly conserved across evolution. 145 Effect of G6PD stabilisers on C. elegans survival in liquid media 4.4.1.1 154 lifespan assay. The G6PD-stabilising small molecule ASW03 improves worm 4.4.1.2 158 survival at 50 µM. 50 µM G6PD-stabilising small molecule ASW08 improves worm 4.4.1.3 160 median survival. Small molecule G6PD stabiliser ASW22 improves worm lifespan 4.4.1.4 161 and healthspan. Small molecule G6PD inhibitor ASW25 has no effect on worm 4.4.1.5 163 survival and healthspan. A single atomic change between G6PD stabilisers ASW06 and 4.4.1.6 166 ASW07 affect compound efficacy in modulating worm lifespan. Antioxidant capacity assessment of lifespan-extending G6PD 4.4.1.7 167 stabilisers. Age-associated decline in pharyngeal pumping ability of C. elegans 4.4.2.1 169 is not improved or rescued by lifespan-improving ASW compounds. ASW compounds improve lifespan of C. elegans but do not prevent 4.4.2.2 or rescue age-induced loss of physical activity measured by 171 thrashing rate. 4.4.3.1 Age-dependent decline of C. elegans G6PD enzyme activity. 173 Eggs within gravid adult C. elegans contributes minimally to the 4.4.3.2 174 overall G6PD activity of whole worms. Lifespan-extending ASW compounds do not improve or rescue age- 4.4.3.3 176 associated loss of G6PD activity. Oxidative stress resulting in worm lethality was induced at 200 mM 4.4.3.4 178 diamide and 100 mM paraquat. ASW compound treated C. elegans exhibited increased resistance to 4.4.3.5 low-dose diamide-induced stress and increased susceptibility to high 180 dose, but not mild paraquat-induced lethality.

Tables Page Clinically important medications implicated in G6PD deficiency 1.3.2 37 haemolysis. Activators of G6PD identified from the HTS G6PD screen 2.4.3.1 89 (Compounds 1-14). Activators of G6PD identified from the HTS G6PD screen 2.4.3.2 90 [continued] (Compounds 15-22). Inhibitors of G6PD identified from the HTS G6PD screen 2.4.3.3 91 (Compounds 23-28). List of primers used for sequence confirmation and site-directed 3.3.3.1 108 mutagenesis 3.3.4.1 Plasmids and their corresponding antibiotics for maintenance. 109 Predicted compound-enzyme binding affinity of ASW compounds 3.4.2.1 120 to G6PD NADP+ binding sites ASW compound interaction with amino acids of G6PD enzyme 3.4.2.2 NADP+ structural binding region determined by computational 123 docking simulations. ASW Compound effect on C. elegans median survival and 4.4.1.1 maximum lifespan in liquid media (Preliminary survival 156 assessment) ASW Compound effect on C. elegans median survival and 4.4.1.2 164 maximum lifespan C. elegans stress resistance assay against diamide and paraquat 4.4.3.1 181 result summary

Supplementary Page Standard curve to determine the absorbance value of converted 1 212 NADP+ and NADPH at 340 nm. Recombinant human wild-type G6PD enzyme activity in the presence 2 213 of enzyme substrates G6P or NADP+. Several small molecules identified from the G6PD HTS do not alter 3 214 enzyme activity. 4 G6PD activators derived from the HTS screen (ASW01 – ASW06). 215 5 G6PD activators derived from the HTS screen (ASW07 – ASW12). 216 6 G6PD activators derived from the HTS screen (ASW13 – ASW18). 217 7 G6PD activators derived from the HTS screen (ASW19 – ASW22). 218 8 G6PD inhibitors derived from the HTS screen (ASW23 – ASW28). 219 Expression of recombinant G6PD enzyme in BL21 (DE3) E. coli in 9 220 the presence or absence of chaperone proteins. 10 Mascot peptide summary report. 221 G6PD/Small molecule interactions determined by Surface Plasmon 11 222 Resonance (SPR). ASW compound treatment on C. elegans survival in liquid media 12 223 survival assay. The G6PD-stabilising small molecule ASW21 improves worm median 13 survival at 50 µM drug dose but may be detrimental to worm 224 maximum lifespan at 10 µM. C. elegans survival assay trialling lead G6PD-stabilising ASW small 14 225 molecules. Experimental repeat #2 of C. elegans survival assay trialling lead 15 226 G6PD-stabilising ASW small molecules. Experimental repeat #3 of C. elegans survival assay trialling lead 16 227 G6PD-stabilising ASW small molecules. Experimental repeat #4 of C. elegans survival assay trialling lead 17 228 G6PD-stabilising ASW small molecules. Experimental repeat #5 of C. elegans survival assay trialling lead 18 229 G6PD-stabilising ASW small molecules.

Abbreviations

6PGD 6-phosphogluconate dehydrogenase ALS Amyotrophic lateral sclerosis ASW Nominated drug term: Ashley Sinclair Wu ATM Ataxia-telangiectasia mutated ATP Adenosine triphosphate AU Absorbance unit BFT Benfotiamine cAMP Cyclic adenosine monophosphate CGC Caenorhabditis Genetics Centre CNSHA Chronic non-spherocytic haemolytic anaemia CUPRAC Cupric reducing antioxidant capacity CuZnSOD Copper-zinc superoxide dismutase DHEA Dehydroepiandrosterone DMSO Dimethyl sulfoxide eNOS Nitric-oxide synthase FuDR 5-fluorouracil-2’-deoxy-ribose G6P Glucose-6-phosphate G6PD Glucose-6-phosphate dehydrogenase GdnHCl Guanidine Hydrochloride GPx Glutathione peroxidase GSH Reduced glutathione GSSG Oxidized glutathione Hb Haemoglobin HGF Hepatocyte growth factor His Histidine HTS High-throughput screen LB Luria Bertani mTOR Mammalian target of rapamycin

NAD Nicotinamide adenine dinucleotide NADH Reduced nicotinamide adenine dinucleotide NADP+ Nicotinamide adenine dinucleotide phosphate NADPH Nicotinamide adenine dinucleotide phosphate hydrogen NAMPT Nicotinyl adenosine monophosphate transferase NGM Nematode growth medium NO Nitric oxide PAINS Pan-assay interfering compounds PARP Poly-ADP-ribose polymerase PKA cAMP-dependent protein kinase A PPP Pentose phosphate pathway PRPP 5-phospho-a-D-ribosyl 1-pyrophosphate R5P Ribose-5-phosphate RBC Red blood cell ROS Reactive oxygen species RPE Ribulose-5-phosphate RPI Ribulose-5-phosphate isomerase S/B Signal to background ratio sHsp Serum heat shock protein SOD1 Copper-zinc superoxide dismutase gene SPR Surface Plasmon Resonance SREBP Sterol-responsive element binding proteins TEV Tobacco Etch Virus TIGAR TP53-induced glycolysis and apoptosis regulator TKT Transketolase TNFα Tumour necrosis factor alpha WHO World Health Organization Za Zoledronic acid

Chapter 1 Introduction and Background Introduction & Background

1.1 The Pentose Phosphate Pathway

Following uptake into cells, glucose undergoes one of three fates after being phosphorylated by hexokinase/glucokinase enzymes. Glucose-6-phosphate (G6P) may undergo glycolysis to produce energy in the form of adenosine triphosphate (ATP) and nicotinamide adenine dinucleotide hydride (NADH), or may be stored as energy in the form of glycogen. Alternatively, G6P may also enter the pentose phosphate pathway (PPP), which runs in parallel to glycolysis (Kletzien, Harris & Foellmi, 1994; Luzzatto, 1986; Stanton, 2012).

1.1.1 Roles and function

The PPP functions to serve as a supply source of pentose intermediates contributing to nucleotide synthesis, catalyse the interconversion of 3-7 carbon sugars for a variety of metabolic purposes, and is a major source of reducing equivalents for the maintenance of the intracellular redox potential (Kletzien, Harris & Foellmi, 1994). This pathway is comprised of two separate branches which are connected: the oxidative branch, which is irreversible, and the non-oxidative branch, which is composed of reactions which are fully reversible. The PPP results in the production of ribose-5- phosphate (R5P) necessary for the synthesis of nucleotides and nucleic acids. It also generates the glycolytic intermediates glyceraldehyde-3-phosphate and fructose-6- phosphate, which may be cycled back into glycolyis, and finally, generate reducing power in the form of nicotinamide adenine dinuclueotide phosphate (NADPH), which is generated from the reduction of NADP+ by the enzyme glucose-6-phosphate dehydrogenase (G6PD), as well as the enzyme 6-phosphogluconate dehydrogenase (6PGD), the next enzyme within the pathway (Figure 1.1.1.1) (Horecker, 2002).

Figure 1.1.1.1 Summary of the Pentose Phosphate Pathway. The Pentose Phosphate Pathway runs in parallel to glycolysis, with the oxidative phase of the PPP producing NADPH from NADP+ and ribulose- 5-phosphate. Ribulose-5-phosphate may be converted to glyceraldehyde-3-phosphate and fructose-6- phosphate which may then be recycled into glycolysis. Xylylose-5-phosphate and Erythrose-4-phosphate which may also be recycled to glycolysis via the enzyme Transketolase (TKT). Abbreviations: G6PD, Glucose-6-phosphate; 6PGD, 6-phosphogluconate dehydrogenase; RPE, ribulose-5-phosphate 3- epimerase; RPI, ribulose-5-phosphate isomerase; TKT, transketolase; NADP+, nicotinamide adenine dinucleotide phosphate; NADPH, nicotinamide adenine dinucleotide phosphate hydrogen (Stanton, 2012).

1.1.2 Sentinel for defence against oxidative stress: NADPH

NADPH plays a crucial role in a broad range of cellular processes, such as serving as a reducing equivalent in a number of processes (Kirsch & De Groot, 2001), detoxification of lipid hydroperoxides and hydrogen peroxides by NADPH-dependent cytochrome P450 enzymes (Iyanagi, 2007; Munro, Girvan & McLean, 2007) and in the generation of nitric oxide (Leopold, Zhang, Scribner, Stanton & Loscalzo, 2003). It is also used for reactive oxygen species (ROS) generation by phagocytes (Kindzelskii, Ueki, Michibata, Chaiworapongsa, Romero & Petty, 2004), synthesis of fatty acids and cholesterol (Horton, Goldstein & Brown, 2002) as well as the maintenance of redox homeostasis, which has been firmly established to be essential for cellular survival (Filosa et al., 2003; Tian et al., 1998). The antioxidant system in most cells relies on the availability of NADPH, with the glutathione system, catalase, and superoxide dismutase being three major components of this system (Sies, 1997; Zhang et al., 2010).

The four main enzymatic sources of NADPH in mammalian cells include G6PD, 6PGD, malic enzyme and isocitrate dehydrogenase. G6PD is however believed to be the primary source of NADPH in the cell, with G6PD-deficient cells exhibiting accelerated senescence attributed to the elevation and accumulation of oxidative DNA damage (Cheng, Ho, Wu & Chiu, 2004; Tian et al., 1998). Mammalian knockouts or animals with severe G6PD deficiency are embryonically lethal (Longo et al., 2002; Pandolfi, Sonati, Rivi, Mason, Grosveld & Luzzatto, 1995; Yang et al., 2013), and G6PD-deficient cells display reduced resistance to oxidative insults, often resulting in cellular senescence or cell death (Abu-Osba, Mallouh & Hann, 1989; Cheng, Ho, Wu & Chiu, 2004; Fico et al., 2004; Filosa et al., 2003; Ho et al., 2000; Lin, Ho, Cheng, You, Yu & Chiu, 2010; Pandolfi, Sonati, Rivi, Mason, Grosveld & Luzzatto, 1995; Tian et al., 1998). As G6PD impairment significantly affects cellular processes that are dependent on the supply of NADPH, it is believed that the other contributors of NADPH alone are not able to produce sufficient NADPH to sustain normal cellular processes, heavily relying on G6PD as the principle source of intracellular NADPH.

NADPH itself is also thought to be a key antioxidant within the mitochondria, not only acting as an indirectly operating antioxidant via the maintenance of reduced glutathione stores which concomitantly re-reduces dehydroascorbate to vitamin C, but also functioning as a direct-acting antioxidant by interacting directly with free radicals to repair biomolecules (Kirsch & De Groot, 2001). ROS in the form of superoxide anion, and its stoichiometric product, hydrogen peroxide, are generated under normal physiological conditions, which are canonically removed by catalases and peroxidases (Fridovich, 1978; Kirkman & Gaetani, 1984). The activities of peroxidases are dependent upon the availability of reduced forms of glutathione or thioredoxin, and results in the oxidation of reduced glutathione (GSH) to oxidized glutathione (GSSG), and of reduced thioredoxin to oxidized thioredoxin, respectively. Tight regulation of this system is imperative to maintain an optimal GSH:GSSG ratio in the cell, which is critical for cell survival (Meister, 1995), and the reducing equivalents of the enzymes responsible for such are supplied by NADPH. Thus, NADPH functions as an indirectly operating antioxidant, being a hydride (hydrogen anion) donor in the regeneration of active antioxidants such as GSH and thioredoxin (Hanschmann, Godoy, Berndt, Hudemann & Lillig, 2013; Kirsch & De Groot, 2001) (Equation 1).

Equation 1: Role of NADPH in cytoprotective action of GSH

GPx 2GSH + H2O2 2H2O + GSSG

NADP+ NADPH + H+

(Kirsch & De Groot, 2001)

Catalase is another antioxidant enzyme responsible for the conversion of hydrogen peroxide to water (Equation 2) and although it does not require NADPH to do so, there is an allosteric binding site on the enzyme for NADPH which allows NADPH to bind to and maintain the catalytic activity of the enzyme (Hillar & Nicholls, 1992; Kirkman & Gaetani, 1984). Thus, NADPH indirectly modulates the anti-oxidative activity of catalase by increasing the lifetime of the catalytically active catalase. 23

Superoxide dismutase, another enzyme responsible for reducing free radical stress, is not affected by the presence of NADPH, and is able to carry out its role converting superoxide to hydrogen peroxide independent of NADPH. However, if the product hydrogen peroxide is not effectively reduced chemically by the NADPH-dependent enzymes catalase or glutathione, the increased levels of hydrogen peroxide can inhibit the activity of superoxide dismutase via a product inhibition mechanism (Gottfredsen, Larsen, Enghild & Petersen, 2013). Therefore, the antioxidant system surrounding superoxide dismutase also indirectly relies on the availability of NADPH.

Equation 2: Cytoprotective action of catalase

catalase

2H2O2 O2 + 2H2O

(Lenzen, 2008)

Furthermore, NADPH is also utilised as a cofactor by nitric-oxide synthase (eNOS) to convert arginine to citrulline and nitric oxide (NO) (Equation 3), as well as maintaining levels of another cofactor tetrahydrobiopterin via de novo synthesis and the dihydrofolate reductase salvage pathway to modulate vascular endothelial growth factor- mediated angiogenesis (Leopold et al., 2003; Leopold, Zhang, Scribner, Stanton & Loscalzo, 2003).

Equation 3: Synthesis of NO:

+ G + L-arginine + NADPH + H + O2 → N -hydroxy-L-arginine + NADP + G + H2O and 2N -hydroxy-L-arginine + NADPH + H + O2 + → 2L-citrulline + NADP + 2H2O + 2NO

(Kindzelskii, Ueki, Michibata, Chaiworapongsa, Romero & Petty, 2004)

Therefore, as G6PD is a major source of NADPH, the enzyme also plays a crucial role in modulating blood pressure via regulating eNOS activity and NO levels. G6PD has also been shown to co-localise with eNOS (Leopold et al., 2003), which may infer another mechanism by which G6PD modulates NO production. Increasing G6PD activity via the use of a vitamin B1 analogue Benfotiamine, which activates the PPP via increasing G6PD activity and transketolase (TKT) also improved recovery following myocardial infarction (Katare, Caporali, Emanueli & Madeddu, 2010). The use of Benfotiamine as a G6PD activating agent is be elaborated in Section 1.5.2 G6PD activating agents.

NADPH oxidases are another family of enzymes dependent on NADPH, and are involved in many crucial physiological processes such as the production of ROS which are important for vascular redox homeostasis (Lassegue & Clempus, 2003), immune defence (Babior, Lambeth & Nauseef, 2002), and the maintenance of multiple redox- sensitive intracellular signalling pathways (Jiang, Zhang & Dusting, 2011). In most tissue types, NADPH oxidase constitutively produces low levels of superoxide anions (Kindzelskii, Ueki, Michibata, Chaiworapongsa, Romero & Petty, 2004; Lassegue & Clempus, 2003) utilising G6PD-derived NADPH as the main fuel source for this process (Gupte et al., 2009; Matsui et al., 2005), as shown according to the following equation:

Equation 4: Superoxide production by NADPH oxidase:

+ + 1/2NADPH + O2 → 1/2NADP + 1/2H + O2-

(Kindzelskii, Ueki, Michibata, Chaiworapongsa, Romero & Petty, 2004)

Superoxide and NO produced from Equations 3 and 4 yields downstream reactive oxygen metabolites and reactive nitrogen intermediates, which are key factors contributing to the host defence against infectious disease (Kindzelskii, Ueki, Michibata, Chaiworapongsa, Romero & Petty, 2004). As the generation of these metabolites rely on NADPH to serve as an electron donor, NADPH plays a crucial role in maintaining host immune defence during infectious diseases and in tissue damage during autoimmune diseases.

1.1.3 Cell growth, proliferation and DNA repair: Ribose-5-phosphate

Another crucial product of the PPP is ribose-5-phosphate (R5P) which is produced by the non-oxidative phase of the PPP (Stanton, 2012). R5P is necessary for the biosynthesis of nucleic acids, constituting the sugar backbone of nucleotides (Beaconsfield, Ginsburg & Jeacock, 1964; Cosentino, Grieco & Costanzo, 2011), or aromatic amino acid synthesis, and may also be converted to glyceraldehyde-3-phosphate and fructose-6-phosphate through a series of aldolases and transketolases to allow for partial redirection or recycling back into glycolysis (Stanton, 2012). In a single step reaction, two of the phosphates of ATP are transferred to R5P, forming 5-phospho-a-D- ribosyl 1-pyrophosphate (PRPP) (Khorana, Fernandes & Kornberg, 1958). This intermediate is required for the biosynthesis of purines, pyrimidines, histidines, tryptophan and most importantly nicotinamide adenine dinucleotide (NAD) via the stress- and nutrient responsive element NAD+ biosynthetic enzyme nicotinyl adenosine monophosphate transferase (NAMPT) (Becker, Raivio & Seegmiller, 1979; Hove- Jensen, 1988; Zerez & Tanaka, 1989).

NAD serves as an essential cofactor in many biochemical reactions. For example, the oxidized form of NAD (NAD+) is required for glucose and fat oxidation, whereas its reduced form (NADH) serves as a substrate in the electron transport chain. NAD is also consumed as a cofactor by enzymes such as poly-ADP-ribose polymerase (PARPs), the cyclic ADP-ribose hydrolase CD38, and notably, the sirtuins, a class of NAD dependent deacylase enzymes (Verdin, 2014). Studies have shown that NAD levels change with age, and that modulating NAD production or utilization can lead to prolonged lifespan and increased healthspan in a sirtuin-dependent manner (Canto et al., 2012; Mouchiroud et al., 2013; Yoshino, Mills, Yoon & Imai, 2011). The activity of the sirtuin enzymes are strictly dependent on NAD+ availability (Verdin, 2014), and these observations suggest that by increasing NAD+ levels, such as via supplementation with NAD precursors, may have potential as a therapy to enhance healthspan and lifespan.

Therapies that increases NAD+ availability or sirtuin activity are beneficial as they can maintain late-life health in mammals through improved insulin sensitivity, decreased inflammation and improved mitochondrial function (Canto et al., 2012; Gomes et al., 2013; Yoshino, Mills, Yoon & Imai, 2011). As the PPP product R5P forms PRPP, which is a precursor of NAD+, activation of PPP may be a means of increasing NAD+ levels, which may lead to increased healthspan and lifespan. Genome instability and oxidative stress are hallmarks of old age, and there is abundant evidence that the sirtuins slow the pace of biological ageing. Sirtuin activity and NAD+ levels decline with old age (Gomes et al, 2013), and perhaps not coincidentally, so does the PPP (Niedermuller, 1986).

Ribose-5-phosphate had been believed to be the ultimate product of the PPP within cells under normal or neoplastic cell growth conditions, as the requirement of R5P for nucleic acid synthesis was necessary to maintain this growth. Supplementation with RNA, which serves as a physiological source of R5P, rescued growth inhibition in cells with inhibited G6PD activity in one study (Dworkin, Gorman, Pashko, Cristofalo & Schwartz, 1986), however, in another fibroblast cell lines in which G6PD activity, and thus the PPP has been inhibited, supplementation of RNA did not rescue or restore cell growth (Tian et al., 1998). It is unknown if these discrepancies are a result of tissue specificity or experimental method, but it is possible that the requirement of the product NADPH exceeds that of R5P during cell growth and proliferation, or that both NADPH and R5P are necessary products of the PPP required for cell growth. Furthermore, there are other sources of R5P outside of the PPP (Tozzi, Camici, Mascia, Sgarrella & Ipata, 2006), thus the requirements of NADPH may be more crucial compared to the needs for R5P.

1.2 Glucose-6-Phosphate Dehydrogenase Enzyme

The G6PD enzyme is the first, and rate-limiting, enzyme of the PPP. This enzyme is responsible for the irreversible entry of glucose-6-phosphate (G6P) into the PPP via catalysing the oxidation of G6P to 6-phosphogluconate, thereby functioning as a key regulatory enzyme to this pathway (Figure 1.1.1.1). The G6PD gene is highly conserved throughout evolution (Au, Gover, Lam & Adams, 2000; Ho, Cheng & Chiu, 2005; Vulliamy, Mason & Luzzatto, 1992), with multiple alignment of five known G6PD amino acid sequences from diverse species showing over 37% identity to the human sequence (Figure 1.2.1), with this value being greater than 48% when comparing G6PD sequences among eukaryotic organisms only (Au et al., 1999). The 37% sequence identity is high enough to indicate a common fold, with comparison of the amino acid sequence of the G6PD enzyme also revealing several conserved lengths of amino acid sequences which will be elaborated in Section 1.2.1 The G6PD gene/protein structure.

Figure 1.2.1 The G6PD enzyme sequence is highly conserved in a variety of species. (A) Amino acid alignment of G6PD homologues in humans (G6PD_HUMAN), mouse (G6PD1_MOUSE), fruit fly (G6PD_DROME), bacteria (G6PD_ECOLI), and nematodes (G6PD_CAEEL). Asterisk (*) denotes fully conserved residues, colon (:) represents strongly similar properties and period (.) represents weakly similar properties. (B) Phylogenetic relationships of G6PD. Sequence alignment and analyses were conducted using Clustal Omega multiple sequence alignment program.

1.2.1 The G6PD gene/protein structure

The gene encoding G6PD is located in the Xq28 telomeric region of the X- chromosome long arm and is flanked on either side by factor VIII coagulant protein and the red/green color pigment gene (Takizawa, Yoneyama, Miwa & Yoshida, 1987; Vulliamy, Mason & Luzzatto, 1992), with the human G6PD gene being 21kb in length, consisting of 13 exons and 12 introns (Takizawa, Yoneyama, Miwa & Yoshida, 1987). The coding sequence of the gene is 1545bp long and was first deduced in 1986 by Persico et. al. (Persico et al., 1986). The proximal 400bp of the human G6PD gene promoter drives the constitutive gene expression, whilst the distal promoter housing cis-elements which accounts for the adaptive regulation of the gene (Ursini, Scalera & Martini, 1990). Following translation, G6PD exists as a single protein subunit of 515 amino acids with a molecular mass of 59 kDa, containing binding sites for the substrates NADP+ and G6P as well as an allosteric modifier binding site for NADP+ that functions to stabilise the enzyme in its active dimeric/tetrameric equilibrium (Au, Gover, Lam & Adams, 2000; Persico et al., 1986). The pH of the environment of the enzyme determines the state of the enzyme, whereby high pH and ionic strength is known to shift the enzyme equilibrium towards forming more dimers, and low pH towards the tetrameric state (Cohen & Rosemeyer, 1969).

Figure 1.2.1.1 Conserved regions in amino acid alignment of G6PD homologues in humans, mouse, fruit fly, bacteria and nematodes. Asterisk (*) denotes fully conserved resides, colon (:) represents strongly similar properties and period (.) represents weakly similar properties. (A) Conserved sequence region GASGDLAKKK of G6PD across five different species. (B) Completely conserved sequence YRIDHYLGKE corresponding to the enzyme substrate binding site.

Multiple alignment of 35 G6PD amino acid sequences revealed that the G6PD enzyme contains a completely conserved octapeptide sequence RIDHYLG/K (residues 198-205; single-letter amino acid code) which corresponds to the enzyme substrate binding site, and contains a lysine residue that has been shown to be essential for enzyme activity (Au et al., 1999; Bautista, Mason & Luzzatto, 1995; Kletzien, Harris & Foellmi, 1994). Comparison of G6PD amino acid sequences across humans, mice, yeast, fruit flies and nematodes revealed this conserved region to be an octapeptide sequence (Figure 1.2.1.1.A). Another conserved region, a modified nucleotide-binding fingerprint, GxxGDLA (residues 42-48; single-letter amino acid code) was also determined from the multiple alignment of 35 known amino acid sequences of G6PD across varying species (Au et al., 1999), with this region having been associated with coenzyme binding (Levy, Vought, Yin & Adams, 1996). This conserved sequence is ten amino acids in length when multiple alignment across five different species were performed (Figure 1.2.1.1.B).

A substantial number of mutations exist in the human G6PD gene, with 140 mutations or combinations of mutations having been described based on biochemical characteristics of the human enzyme (Beutler, 1991; Beutler & Vulliamy, 2002), with new variants still being discovered recently (Mella et al., 2018). These mutations are widespread across the gene, with only one cluster of mutations emerging between amino acids 380 and 410 of the G6PD gene corresponding to the subunit binding interface of the active G6PD homodimer (Naylor et al., 1996). Nearly all the mutations situated within this cluster present as severe G6PD deficiency Class I phenotype (disease classification is elaborated in Section 1.3.1 Epidemiology, variants and WHO classification), with 34% of class I G6PD deficiency variants being present in this region which represents only 6% of the entire G6PD sequence (Notaro, Afolayan & Luzzatto, 2000). Features and phenotypes resulting from such mutations will be elaborated in the section 1.3 Glucose- 6-phosphate Deficiency.

1.2.2 Positive regulation of G6PD Enzyme Activity

G6PD is a critical metabolic enzyme at the centre of an essential metabolic nexus that affects many physiological processes. Traditionally thought to be under heavy regulation by the NADPH/NADP+ ratio (Holten, Procsal & Chang, 1976; Kletzien, Harris & Foellmi, 1994), whereby a decrease in NADPH/NADP+ ratio increases G6PD activity to replenish NADPH, we now understand that the regulation of the G6PD enzyme is much more complex than previously believed. Adaptive regulation of this enzyme occurs at the level of transcription, translation, intracellular localisation, tissue expression and post- translational modifications of the enzyme. There are many known endogenous positive regulators of G6PD, and subsequently the PPP, which includes vitamin D (Bao, Ting, Hsu & Lee, 2008), insulin, PI3-K, AKT, mTOR (mammalian target of rapamycin) and S6 Kinase (Duvel et al., 2010; Gupte et al., 2009; Wagle, Jivraj, Garlock & Stapleton, 1998) with the list of positive regulators expanding with time.

Dietary carbohydrate and lipid consumption modulates G6PD expression at the enzyme transcriptional (Iritani, 1992; Katsurada, Iritani, Fukuda, Matsumura, Noguchi & Tanaka, 1989) and translational level (Katsurada, Iritani, Fukuda, Matsumura, Noguchi & Tanaka, 1989; Tomlinson, Nakayama & Holten, 1988), to support the increased need for NADPH for the synthesis of fatty acids in cells. Insulin has been shown to be involved in this regulation, first being demonstrated by Kurtz and Wells (Kurtz & Wells, 1981) in primary cultures of hepatocytes, with similar observations being made in other studies (Stumpo & Kletzien, 1984). This insulin-induced activation of G6PD occurs downstream of phosphatydylinositol-3 kinase and the serine/threonine kinase AKT (Wagle, Jivraj, Garlock & Stapleton, 1998). The G6pd gene is also regulated at the transcriptional level based on the increased need for NADPH during the biosynthesis of lipids via SREBPs (Sterol-responsive element binding protein), which are major lipid metabolism regulating transcription factors (Horton, 2002), as well as by transcription factor Nrf2, which is responsible for the regulation of several antioxidant response elements genes (Zhang & Wang, 2007). The expression of G6PD is also regulated by ROS, with G6PD mRNA levels being elevated in the presence of increased oxidative stress (Cramer, Cooke, Ginsberg, Kletzien, Stapleton & Ulrich, 1995).

Serum heat shock proteins, such as sHsp27, are also known to modulate and induce G6PD activation on a post-translational basis, which allows for the maintenance of GSH in its reduced state during oxidative stress in a G6PD-dependant manner (Preville et al., 1999). sHsp27 was shown to directly increase G6PD activity in vitro, possibly by stabilising the G6PD protein in its active configuration (Cosentino, Grieco & Costanzo, 2011) and by associating with the ATM protein which then maintains the G6PD protein. The tumour suppressor p53, which is known to repress G6PD, also stimulates an activator of G6PD known as TIGAR (TP53-induced glycolysis and apoptosis regulator) (Bensaad et al., 2006; Jiang et al., 2011), which has an ability to increase the flux of PPP to assist in the elimination of ROS, creating a tight regulation of G6PD activity. The active form of vitamin D, 1α, 25- dihydroxy-vitamin D3 had also been reported to confer resistance of non-malignant cells towards oxidative stress-induced cell death in a G6PD-dependent manner via reduction of ROS-induced cellular stress and modulation of G6PD in a transcriptional manner (Bao, Ting, Hsu & Lee, 2008).

1.2.3 Negative regulation of G6PD Enzyme

G6PD activity and expression is also regulated by negative regulators such as cyclic adenosine monophosphate (cAMP) and cAMP-dependent protein kinase A (PKA), which plays a crucial role in the tight coupling between NADPH production and utilization by enzymes such as NADPH oxidase in macrophages during inflammation (Costa Rosa, Curi, Murphy & Newsholme, 1995). At the transcriptional level, cAMP- response element modulator decreases the transcription of the G6PD gene (Leopold et al., 2007), and PKA increases serine/threonine phosphorylation of G6PD leading to enzyme downregulation (Xu, Osborne & Stanton, 2005).

Tumour necrosis factor alpha (TNFα) has also been shown to augment G6PD activity. The regulatory effect of TNFα on the expression and activity of G6PD remains to be elucidated, with both evidence suggesting a positive regulatory effect (Spolarics & Wu, 1997), and negative regulatory effect (Zhang & Wang, 2007) of the cytokine being present in the literature. The tumour suppressor p53 is also capable of regulating G6PD activity. This protein is frequently mutated in tumours and is capable of interacting transiently with the G6PD enzyme to prevent the formation of active dimers (Jiang et al., 2011), thereby inhibiting G6PD activity and reducing the PPP shunt. The inhibitory effect of p53 on the PPP may be a feature of the anti-proliferative role of the p53 gene, allowing control over glucose biosynthesis and catabolism which is elevated in cancer cells (DeBerardinis, Lum, Hatzivassiliou & Thompson, 2008). The interaction of multiple signals as mentioned above ultimately results in the final cellular phenotype, with G6PD being in the midst of a complex metabolic nexus.

The G6PD enzyme is also regulated post-translationally, with acetylation of the G6PD enzyme resulting in enzyme inactivation crucial for tight regulation of NADPH homeostasis (Wang et al., 2014). An example of a protein deacetylase is the SIRT2 enzyme, which relies on the availability of cofactor NAD+ for action which is a downstream product of the PPP as described in Section 1.1.3, plays a crucial role in G6PD deacetylation and activation, creating a feedback loop between G6PD and the sirtuin enzyme (Wang et al., 2014).

1.3 Glucose-6-phosphate Dehydrogenase Deficiency

Having first been described by Warburg and Christian in 1931 (Kornberg, Horecker, Horecker & Smyrniotis, 1955), the enzyme G6PD has garnered interest in regards to G6PD deficiency and its associated haemolysis (Beutler, 1994; Luzzatto, 2006; WHO, 1989), as well as its involvement in lipid metabolism (Nkhoma, Poole, Vannappagari, Hall & Beutler, 2009). The genetic variant of human G6PD is the most common and heterogenous human enzymopathy (Beutler & Vulliamy, 2002), with an estimated 330-400 million affected, or around 4.9% of the global population (Cappellini & Fiorelli, 2008; Nkhoma, Poole, Vannappagari, Hall & Beutler, 2009; WHO, 1989). The G6PD gene is unique amongst housekeeping genes because of the many human mutations which have been discovered, many of which are polymorphic rather than sporadic (Notaro, Afolayan & Luzzatto, 2000). As a result of the prevalence of this disease, the gene sequence, protein sequence, and crystal structure of the G6PD enzyme variants have been extensively researched (Au, Gover, Lam & Adams, 2000).

G6PD deficiency is the result of an array of mutations affecting the coding sequence of the G6PD gene (Persico et al., 1986; Takizawa, Yoneyama, Miwa & Yoshida, 1987). The majority of the mutations are missense mutations, with early nonsense or ‘null’ mutations, mutations causing destruction of the reading frame or mutations causing amino acid substitutions (Beutler & Vulliamy, 2002) resulting in rapid enzyme degradation due to enzyme instability and/or reduced affinity towards the enzyme’s substrates. Mutations affecting substrate or coenzyme binding regions however, have never been observed (Luzzatto, 2006; Naylor et al., 1996). The mutations are also widespread across the G6PD gene sequence with only one discrete cluster present within the amino acid range 380-410 (Notaro, Afolayan & Luzzatto, 2000), corresponding to the subunit interface in an enzymatically active G6PD dimer (Naylor et al., 1996). No mutations within the fully conserved amino acids regions have been reported (as described in Section 1.2.1 The G6PD gene/protein structure; Figure 1.2.1.1), presumably due to the importance of these regions for the maintenance of enzyme function and activity, with replacement of these residues having a high probability of being lethal at an early stage of development or life (Notaro, Afolayan & Luzzatto, 2000).

1.3.1 Epidemiology, Variants and WHO classification

G6PD deficiency is widespread with variants of the gene prevailing in certain regions. In some villages in Sardinia over 30% of male inhabitants have G6PD deficiency (Siniscalco, Bernini, Latte & Motulsky, 1961), with the deficiency being observed in 13% of Saudis (Gelpi, 1967), and 11% of black Americans (Heller, Best, Nelson & Becktel, 1979). The prevalence of G6PD deficiency had also been reported to be 2 – 16% in Taiwan and Southern China in a study performed in 1988 (Du, Xu, Hua, Wu & Liu, 1988), with G6PD Canton being one of the most common deficient variant in east Asians, having a gene frequency reported reaching up to 1.7% of the population in Southern China (McCurdy, Kirkman, Naiman, Jim & Pickard; Stevens, Wanachiwanawin, Mason, Vulliamy & Luzzatto, 1990). G6PD Mahidol is one of the most common deficiency variants in South East Asia, with the prevalence of this deficiency having been reported as affecting as many as 15% of the population in some areas (Vulliamy, Wanachiwanawin, Mason & Luzzatto, 1989), whilst G6PD Mediterranean is a single widespread mutation which accounts for G6PD deficiency in 70% of male Kurdish Jews (Oppenheim, Jury, Rund, Vulliamy & Luzzatto, 1993). The observed widespread dispersion of the deficiency is believed to be attributed to the conferred protection against Falciparum malaria, with a near complete overlap between malaria endemic region and the distribution of the deficiency (Luzzatto & Notaro, 2001; Mbanefo et al., 2017; Vulliamy, Mason & Luzzatto, 1992).

The World Health Organization (WHO) classifies all biochemical variants of G6PD into five groups based on enzyme activity as well as the clinical manifestations exhibited by affected individuals (WHO, 1989). Class I includes severely deficient variants that exhibit less than 1% residual enzyme activity, and results in chronic non- spherocytic haemolytic anaemia (CNSHA). Class II mutants exhibit 1-10% of normal enzyme activity but are not accompanied by CNSHA. Class III variants are characterized by having mild to moderate enzyme activity (10-60% activity), Class IV variants have normal enzyme activity and Class V variants exhibit greater than normal enzyme activity (Cappellini & Fiorelli, 2008; WHO, 1989). Most Class I mutations, which are characterised as having low (<1%) enzyme activity and thermostability, are located at the dimeric interface of the enzyme (Naylor et al., 1996), weakening the dimerization of the G6PD enzyme and resulting in impaired enzyme activity. 35

1.3.2 Phenotype and clinical manifestations

Individuals suffering from this deficiency exhibit phenotypes varying from negligible to severe, depending on whether one or both alleles are affected, as well as the nature of the mutation (Mehta, 1994). Affected individuals are often asymptomatic, but may also experience episodic haemolytic anaemia following exposure triggering agents such as sulphur based drugs, oxidative stress, consumption of fava beans, exposure to various drug compounds such as naphthalene (moth balls), and even infection resulting in life-long haemolytic anaemia (Beutler, 1991). The deficiency also predisposes individuals to symptoms such as neonatal jaundice, and the less common chronic non- spherocytic haemolytic anaemia. The most devastating potential outcome of this deficiency in newborn is an acute haemolytic crisis, causing extreme hyperbilirubinemia which may result in acute bilirubin encephalopathy (Kaplan & Hammerman, 2010).

Most cells have a series of directly-acting antioxidants to combat endogenously produced free radicals, such as the enzymes catalase and superoxide dismutase, as well as vitamin E, which detoxifies hydrophilic regions of the cell (Birben, Sahiner, Sackesen, Erzurum & Kalayci, 2012). Human erythrocytes however rely on G6PD and the antioxidative function of NADPH to combat free radicals, as erythrocytes are devoid of cellular organelles and do not have alternative sources of NADPH (Kirkman & Gaetani, 1986; Luzzatto, 1967) while constantly being exposed to oxidative stress generated during oxidation of haemoglobin (Winterbourn, Benatti & De Flora, 1986). This combination of the generation of deleterious oxidative species and heavy reliance on NADPH produced by the G6PD enzyme to combat free radicals results in premature cell red blood cell lysis in G6PD deficiency patients when exposed to chemicals or drugs which are known haemolytic agents (Wajcman & Galacteros, 2004). The disease presents itself even if a person is heterozygous for G6PD deficiency, as haemolysis of 50% of red blood cell is sufficient to release enough haemoglobin (Hb) to be catabolised to bilirubin, resulting in hyperbilirubinemia (Kaplan & Hammerman, 2010).

Table 1.3.2 Clinically important medications implicated in G6PD deficiency haemolysis.

Well established clinically important medications implicated in G6PD deficiency haemolysis Medication/chemical Purpose/use Reference 1 Primaquine 8-aminoquinoline anti-malarial (Ashley, Recht & White, 2014; Beutler, Dern & Alving, 1954; Youngster et al., 2010) (Chan, Todd & Tso, 1976) 1 Dapsone Sulfone anti-microbial used in the treatment (Degowin, Eppes, Powell & of leprosy and pneumonia, prophylaxis Carson, 1966; Fanello et al., 2009) 1 Rasburicase Recombinant urate oxidase enzyme used in (Browning & Kruse, 2005) the treatment of hyperuricemia (Sherwood, Paschal & Adamski, 2016) 1 Methylene blue Treatment of methemoglobinemia (Beutler, 1994; Youngster et al., 2010) 1 Toluidine blue Used in the detection of oral and thyroid (Teunis, Leftwich & Pierce, malignancies 1970; Youngster et al., 2010) 1 Phenazopyridine Urinary tract analgesic used in the treatment (Galun, Oren, Glikson, of dysuria Friedlander & Heyman, 1987; Mercieca, Clarke, Phillips & Curtis, 1982) 1 Nitrofurantoin Anti-bacterial used in the treatment of (Lavelle, Atkinson & Kleit, urinary tract infections 1976) (Chan, Todd & Tso, 1976) 1 Pegloticase Used in the prevention of gout (Geraldino-Pardilla, Sung, Xu, Shirazi, Hod & Francis, 2014; Owens, Swanson & Twilla, 2016) 1 Sulfonamides Anti-microbial/antibiotics/anti- (Beutler, 1991; Chan, Todd & inflammatory Tso, 1976; Prankerd, 1963) 2 Acetylsalicylic acid Analgesic and antipyretic (Chan, Todd & Tso, 1976; Feghaly, Al Hout & Mercieca Balbi, 2017; Glader, 1976) 2 Ascorbic acid Nutritive agent and urinary acidifier (Beutler, 1994; Quinn, Gerber, Fouche, Kenyon, Blom & Muthukanagaraj, 2017; Rees, Kelsey & Richards, 1993; Udomratn, Steinberg, Campbell & Oelshlegel, 1977) 2 Paracetamol Analgesic (Cottafava, Nieri, Franzone, Sanguinetti, Bertolazzi & Ravera, 1990; Sklar, 2002; Wright, Perry, Woolf & Shannon, 1996)

1 Medications in clinical use that should be avoided with G6PD deficiency; 2 Medications which may exacerbate symptoms, but overall evidence does not suggest against use with G6PD deficiency. Drugs with insufficient evidence towards G6PD deficiency were omitted (Belfield & Tichy, 2018; Youngster et al., 2010).

Drugs that are considered to be haemolytic agents include anti-malarials such as primaquine and pamaquine, sulphonamides and sulphones, analgesics such as acetylsalicylic acid (aspirin) and some anti-bacterial compounds (WHO, 1989), with other clinically relevant drugs known to be haemolytic agents being summarised in Table 1.3.2. Following exposure to a haemolytic agent, G6PD-deficient individuals may be asymptomatic, or may express the disease ranging from mild haemolytic anaemia, haemolytic crisis and hereditary non-spherocytic haemolytic anaemia, to sepsis, neonatal jaundice and life-threatening kernicterus in newborns (Abu-Osba, Mallouh & Hann, 1989; Meloni, Cutillo, Testa & Luzzatto, 1987). Other triggering agents that are consistently associated with haemolysis includes fava beans or food cross-contaminated with fava beans (Beutler, 1994; Zuccotti et al., 2014), naphthalene (Santucci & Shah, 2000), Henna (Lawsonia inermis) (Kok, Ertekin, Ertekin & Avci, 2004; Raupp, Hassan, Varughese & Kristiansson, 2001) and 1-phenylazo-2-naphthol-6-sulfonic acid, found in food coloring agent Orange-RN (Akinyanju & Odusote, 1983) resulting in the food additive being banned from use in many countries (EU directive 76/399/EEC). Several other reported triggers of haemolysis includes traditional Chinese medicine, herbs and Ayurvedic medicine but with insufficient evidence linking the agents to haemolytic anaemia in G6PD deficiency (Lee, Lai, Chaiyakunapruk & Chong, 2017).

G6PD-deficient heterozygous women also experience an increased rate of spontaneous abortions in their first trimester when compared to normal G6PD women (Toncheva & Tzoneva, 1985), suggesting that G6PD may play an important role in fertility and pregnancy. The importance of G6PD in maintaining a successful pregnancy was also further supported with mice exhibiting reduced G6PD activity yielding smaller litter sizes (Nicol, Zielenski, Tsui & Wells, 2000; Tsai, Schulte, O'Neill, Chi, Frolova & Moley, 2013) and G6PD-knockdown in the nematode Caenorhabditis elegans resulting in defective oogenesis and reduced egg production (Yang et al., 2013). In an analysis from 1992-2004 (Registry for newborn Kernicterus), 4 out of 26 (~15%) infants with confirmed G6PD enzyme deficiency died during their first year with 8 out of the 26 (~31%) having concurrent sepsis followed by 2 deaths (Johnson, Bhutani, Karp, Sivieri & Shapiro, 2009). Furthermore, higher total oxidative stress with decreased G6PD activity is associated with poorer sperm motility and quality which may also contribute to impairment human fertility (Dobrakowski, Kasperczyk, Horak, Chyra-Jach, Birkner & Kasperczyk, 2017). 38

G6PD deficiency has also been hypothesised to be beneficial in regards to the reduction of reductive stress (Rajasekaran et al., 2007). Reductive stress is defined as an abnormal increase in reducing equivalents such as GSH and NADPH, which may result in molecular pathogeneses such as increased ROS production, protein misfolding and aggregation, mitochondrial dysfunction, and nitrosative stress (Bukau, Weissman & Horwich, 2006; Dimmeler & Zeiher, 2007; Shao, Oka, Brady, Haendeler, Eaton & Sadoshima, 2012; Tome, Johnson, Samulitis, Dorr & Briehl, 2006; Zhang et al., 2012). There are also multiple studies showing that decreasing G6PD activity was useful in alleviating reductive stress (Dimmeler & Zeiher, 2007; Gupte et al., 2009). Reduced G6PD activity could however still have adverse effects if antioxidant defences are lowered (Hecker et al., 2013; Jain et al., 2003). G6PD deficiency also confers a protective role against infection with Plasmodium falciparum malaria, attributing to the observed near complete overlap between the dispersion of G6PD deficiency and the endemic malaria (Luzzatto & Notaro, 2001) caused by increased phagocytosis of the infected erythrocyte (Cappadoro et al., 1998).

There have also been cases of G6PD deficiency resulting in chronic and severe haemolytic anaemia as opposed to the more common acute, sporadic haemolysis, with leucocytes being affected, causing impairment in leucocyte function and decreased capacity to combat infections (Gray et al., 1973). Infections are associated to the symptoms of G6PD deficiency, noted to be a common trigger of haemolysis in G6PD- deficient patients hypothesised to be caused by the release of ROS by leucocytes during phagocytosis resulting in oxidative stress to the erythrocytes (Beutler, 1994; Cappellini & Fiorelli, 2008; Frank, 2005). Notable infections which induce haemolytic anaemia in G6PD deficient individuals includes pneumonia (Tugwell, 1973), hepatitis viruses A and B (Bandyopadhyay et al., 2015; Gotsman & Muszkat, 2001; Kattamis & Tjortjatou, 1970).

1.3.3 Management of G6PD deficiency

Most individuals with G6PD deficiency are asymptomatic in the absence of exposure to haemolytic agents such as fava beans (Beutler, 1994), naphthalene (moth balls) (Santucci & Shah, 2000) as well as several clinically relevant medications as described in Table 1.3.2 in Section 1.3.2 Phenotype and clinical manifestations. In the presence of these oxidative triggers however, affected individuals may experience acute haemolytic anaemia accompanied with malaise, neonatal jaundice or hyperbilirubinemia (Beutler, 1991; Kaplan & Hammerman, 2010). As such, avoidance of triggering agents is the main recommended solution for the management of G6PD deficiency with no other interventions required. Complete avoidance however may not always be possible and may present problems particularly with avoidance of pharmaceuticals. An example of medical avoidance impacting on the treatment of diseases not related to G6PD deficiency is the avoidance of anti-malarials which are known haemolytic agents necessary for the treatment and management of malaria.

Primaquine is an 8-aminoquinoline, currently the only available drug effective for the treatment of malaria and is prescribed to prevent the transmission of malaria in endemic countries, with the drug proven effective in the eradication of liver hypnozoites of Plasmodium vivax and Plasmodium ovale and for use in the prevention or relapse of the disease (Jalloh et al., 2004). Anti-malarial 8-aminoquinoline compounds such as primaquine and pamaquine are well documented haemolytic agents which exacerbate the symptoms of G6PD deficiency (Ashley, Recht & White, 2014; Beutler, Dern & Alving, 1954; Howes, Battle, Satyagraha, Baird & Hay, 2013). The susceptibility of G6PD- deficient individuals to anti-malarials are a problem of public health importance, as there is an almost complete geographic overlap in regions of increased incidence of G6PD deficiency with those of which incidences of malarial infection remains prevalent (Luzzatto & Notaro, 2001), which is widely believed to have evolved because of selective pressures exerted by Plasmodium falciparum malaria (Vulliamy, Mason & Luzzatto, 1992).

Reduced antioxidant capacity via impaired glutathione regeneration is a possible adverse effect of G6PD deficiency (Jilani & Iqbal, 2011), with impaired antioxidative function contributing to shortened red cell survival (Abdul-Razzak, Nusier, Obediat & Salim, 2007; Gong, Tian, Huang, Wang & Xu, 2017). Vitamin E, or α-tocopherol is a lipophilic antioxidant that reacts with free radicals (Cadenas, 1989), and is known to prevent red blood cell (RBC) lysis in vitro following the induction of oxidative stress (Corash et al., 1980; Rose & Gyorgy, 1950). Administration of vitamin E had been debated as a potential therapy for the management of G6PD deficiency, with several studies suggesting improved patient outcome following haemolytic episodes (Corash et al., 1980; Eldamhougy, Elhelw, Yamamah, Hussein, Fayyad & Fawzy, 1988; Hafez et al., 1986; Newman, Newman, Bowie & Mendelsohn, 1979; Sultana et al., 2008), while others suggesting against this (Johnson, Vatassery, Finkel & Allen, 1983; Newman, Newman, Bowie & Mendelsohn, 1979). The difference in the findings could be attributed to the small sample size conducted in these studies, as well as antioxidant vitamin deficiency often being co-exhibited in G6PD deficient patients (Jilani & Iqbal, 2011).

Non-life threatening episodic anaemia are also typically managed with regular supplementation of folic acid (Luzzatto, 2006) or xylitol administration accompanied with haematological surveillance, with xylitol administration restoring and maintaining GSH in erythrocytes and conferring protection against induced haemolytic anaemia (Wang, Patterson & Van Eys, 1971). Despite the potential use of xylitol as a treatment for G6PD deficiency, unpublished reports and observations of toxicity in humans following infusions of xylitol ceased any further exploration (Wang, Patterson & Van Eys, 1971). Treatment through haemodialysis also leads to increased G6PD activity and improvements in anaemia of G6PD deficient patients (Ayesh Haj Yousef, Bataineh, Elamin, Khader, Alawneh & Rababah, 2014) thus making blood transfusions a recommended treatment in cases of life-threatening haemolytic crisis and acute renal failure (Beutler, 1994; Luzzatto, 2006).

1.4 The Pentose Phosphate Pathway in Ageing and pathophysiology

The accumulation of ROS oxidative damage during life has been proposed to play a key role in the development of age-dependent diseases such as cardiovascular diseases, cancer as well as ageing (Ames, Shigenaga & Hagen, 1993; Valko, Rhodes, Moncol, Izakovic & Mazur, 2006), with ROS believed to be involved directly or indirectly with the development of cancer (Hercberg, Galan, Preziosi, Alfarez & Vazquez, 1998). Many defence mechanisms have evolved to limit the damage inflicted by ROS in the body, such as the glutathione system, catalase and superoxide dismutase, all of which forms the nexus of the antioxidative system and relies on the availability of the substrate NADPH (Sies, 1997; Zhang et al., 2010). As the PPP is the main source of NADPH and is involved in the maintenance of redox homeostasis and the antioxidative defence (Filosa et al., 2003; Tian et al., 1998; Zhang et al., 2010), as described in more detail in Section 1.1.2 Sentinel for defence against oxidative stress, the G6PD enzyme is a potential therapeutic target for the treatment or management of ROS-associated diseases related to ageing. The role of the G6PD enzyme in pathophysiological diseases related to ageing are elaborated in sections below.

1.4.1 Diabetic phenotype and nephropathy

Diabetes is a common endocrine disorder caused by defects in insulin secretion and/or insulin action caused by chronic hyperglycaemia with disturbances of carbohydrate, fat and protein metabolism (Alberti & Zimmet, 1998). There are many studies proposing a link between having low G6PD activity in blood and the development of diabetes and renal failure (Carette, Dubois-Laforgue, Gautier & Timsit, 2011; Heymann, Cohen & Chodick, 2012; Lai, Lai & Lee, 2017; Niazi, 1991; Pinna, Contini, Carru & Solinas, 2013; Saeed, Hamamy & Alwan, 1985; Wan, Tsai & Chiu, 2002), with diabetic animals also exhibiting decreased G6PD activity in several tissues such as the liver, heart and kidney (Diaz-Flores et al., 2006; Katare, Caporali, Emanueli & Madeddu, 2010; Xu, Osborne & Stanton, 2005). Cardiac progenitor cells isolated from diabetic mice also exhibited reduced G6PD activity (Katare et al., 2013), whilst G6PD activity remains unchanged or even elevated in other tissues under hyperglycaemic conditions such as the brain, lung and heart (Gok et al., 2016; Serpillon et al., 2009; Ulusu et al., 2017), which may be pro-inflammatory or pro-oxidant, elucidating tissue-specific alterations of the G6PD activity in diabetic state.

The hyperglycaemic state of diabetic patients is also believed to be contributing to the further decline of G6PD activity (Carette, Dubois-Laforgue, Gautier & Timsit, 2011; Zhang, Apse, Pang & Stanton, 2000), creating a hypothesis whereby G6PD deficiency may be a risk factor for the development of diabetes. This decrease in G6PD activity has been shown to be inversely correlated with increasing urine albumin levels, protein kinase C activity, and increased nuclear factor-κB activity, suggesting that reduced G6PD activity may contribute to kidney cell damage, with kidney cells also showing elevated markers of inflammation and proapoptotic proteins (Xu et al., 2010) resembling the characteristics seen in diabetic mice. The observed pathogenesis is likely to be caused by the impaired antioxidant system resulting from impaired G6PD activity leading to oxidative stress caused by the exposure to high glucose (Xu, Osborne & Stanton, 2005; Zhang, Apse, Pang & Stanton, 2000), in combination with the vulnerability of pancreatic β cells to the cytotoxic action of ROS because of their low levels of antioxidative enzymes (Tiedge, Lortz, Drinkgern & Lenzen, 1997). Population- wide studies also indicated increased prevalence of diabetic patients with G6PD

43 deficiency compared to the background rate of the general population (Carette, Dubois- Laforgue, Gautier & Timsit, 2011), further supporting the hypothesis.

Increased oxidant production and/or reduced antioxidant capacity has long been characterised as a leading contributor towards the onset of diabetic nephropathy (Kashihara, Haruna, Kondeti & Kanwar, 2010), with extensive studies elucidating oxidative stress to play a significant role in the complications of diabetes mellitus (Carette, Dubois-Laforgue, Gautier & Timsit, 2011; Zhang et al., 2010). This shift in redox balance occurs due to the reduction of G6PD activity resulting from increased glucose-induced activation of PKA leading to the subsequent phosphorylation and inhibition of the enzyme, ultimately resulting in impaired NADPH supply (Zhang, Apse, Pang & Stanton, 2000). Pancreatic β-cells, which are highly sensitive to ROS damage (Robertson, Tanaka, Takahashi, Tran & Harmon, 2005) due to their low levels of the antioxidative enzymes glutathione peroxidase, catalase, and superoxide dismutase (Lenzen, 2008; Lenzen, Drinkgern & Tiedge, 1996; Tiedge, Lortz, Drinkgern & Lenzen, 1997), rely on this NADPH source to combat against oxidative stress. The high glucose concentration observed in diabetic patients reduces G6PD activity in these β-cells, resulting in a decrease in NADPH and ultimately cell death, leading to an overt diabetic phenotype, with over-expression of G6PD alleviating these observed effects (Zhang et al., 2010), further reinforcing the role of G6PD in the development of diabetes. Furthermore, inhibiting PKA activity to increase G6PD activity via the use of hepatocyte growth factor (HGF) rescues kidney mesangial cells from oxidative stress, reinforcing this hypothesis (Hui, Hong, Jingjing, Yuan, Qi & Nong, 2010). The pathogenesis of anaemia observed in patients suffering from end-stage renal disease is also likely caused by the decreased G6PD particularly in the erythrocytes of patients resulting in haemolysis (Chauhan, Gupta, Nampoothiri, Singhai, Chugh & Nair, 1982; Locatelli, Canaud, Eckardt, Stenvinkel, Wanner & Zoccali, 2003).

1.4.2 G6PD expression in cancer

Over-expression or elevated levels of G6PD have been well described in many cancers (Boros, Brandes, Yusuf, Cascante, Williams & Schirmer, 1998; Dessi, Batetta, Pani, Barra, Miranda & Puxeddu, 1990; Frederiks, Vizan, Bosch, Vreeling-Sindelarova, Boren & Cascante, 2008; Jiang, Du & Wu, 2014; Kowalik, Columbano & Perra, 2017; Sulis, 1972) resulting in increased resistance to cell death (Hu et al., 2013), and over- expression of G6PD initiating tumours in nude mice (Kuo, Lin & Tang, 2000). Tumours of G6PD-deficient patients also characteristically exhibit elevated G6PD levels compared to other tissue types (Cocco, Dessi, Avataneo, Picchiri & Heinemann, 1989). This is believed to occur due to an increase in NADPH, which is believed to result in tumour growth (Kuo, Lin & Tang, 2000) as well as an increase in R5P which supports rapid DNA synthesis in proliferating cancerous cells (Boros, Brandes, Yusuf, Cascante, Williams & Schirmer, 1998). The use of G6PD inhibitors such as dehydroepiandrosterone (described in detail in Section 1.5.1 G6PD inhibiting agents) also preferentially inhibited the growth and proliferation of cancerous cells which has high demand for metabolites (Lopez-Marure, Contreras & Dillon, 2011; Simile et al., 1995).

It is also well established that G6PD activity is elevated under proliferative conditions of normal tissue (Molero, Benito & Lorenzo, 1994), but reduced expression of G6PD resulted in better survival and reduced rates of metastasis in cancer patients (Debeb et al., 2016). Correlation between G6PD deficiency and the risk of cancer development remains to be elucidated with evidence suggesting protection against carcinogenesis in G6PD-deficient patients (Dore, Davoli, Longo, Marras & Pes, 2016; Sulis, 1972), others exhibiting no association between G6PD deficiency and cancer progression risk (Cocco, Dessi, Avataneo, Picchiri & Heinemann, 1989; Ferraris, Broccia, Meloni, Forteleoni & Gaetani, 1988) and a recent study suggesting an increased risk of cancer development in G6PD-deficient patients (Yang, Huang, Lin & Chang, 2018). The discrepancies in the observation of cancer development with G6PD deficiency may lie in the difference in pathogenesis and type of the cancer. The PPP can feed back into glycolysis, via the activity of the enzyme transketolase (TKT), which is the rate limiting enzyme catalysing several reactions in the non-oxidative branch of the PPP (Figure 1.1.1.1) and serves as a reversible link between the PPP and glycolysis. This increased activity of TKT may allow cancer cells to adapt to altered environmental conditions and metabolic needs, with TKT

45 regulating the ribose pool (Frederiks, Vizan, Bosch, Vreeling-Sindelarova, Boren & Cascante, 2008; Horecker, 2002; Ramos-Montoya et al., 2006).

Current cancer therapies such as chemotherapy functions by significantly inducing oxidative stress in cancer cells, with increased antioxidative capacity following elevation of the PPP in aberrant cells conferring resistance against the induced stress caused by the treatment (Ramanathan, Jan, Chen, Hour, Yu & Pu, 2005). It is therefore widely believed that PPP inhibition and subsequently impairment of the antioxidative capacity of cancer cells may be an attractive therapeutic strategy against cancer (Jones & Schulze, 2012; Mele et al., 2018). Inhibition of PPP proteins, particularly the G6PD protein has proved beneficial in inducing cell death and suppressing the growth of cancer cells in ovarian cancer (Catanzaro et al., 2015), breast cancer (Li, Fath, Scarbrough, Watson & Spitz, 2015) and urinary bladder cancer (Wang et al., 2015). That being said, overproduction of ROS through either endogenous or exogenous sources induces DNA damage, and the accumulation of this DNA damage may result in multistep carcinogenesis (Valko, Rhodes, Moncol, Izakovic & Mazur, 2006). As G6PD plays an important role in mediating antioxidant defence, an increase in activity of the enzyme may still have a beneficial effect in cancer prevention. It is hypothesised that combined treatments resulting in the upregulation of G6PD activity and decrease in transketolase activity via the use of inhibitors could be an effective method to induce apoptosis in cancer cells via the increased production in NADPH with a simultaneous decrease in reversible carbon interchange intermediates (Boros, Brandes, Yusuf, Cascante, Williams & Schirmer, 1998).

1.4.3 Neurodegenerative diseases

Age-related deterioration of neuronal tissues are primarily caused by destructive free radicals naturally produced during aerobic metabolism, with the combination of high metabolism and the non-replicating nature of neuronal cells increasing the vulnerability of the central nervous system to developing irreversible and cumulative damage (Merlo et al., 2005; Reiter, 1995) which may be causative in the development of neurodegenerative disorders such as Alzheimer’s, Parkinson’s and Huntington’s disease (Behl & Moosmann, 2002; Halliwell, 2001; Markesbery & Carney, 1999). The brain is highly dependent on the antioxidative enzymes to reduce and combat oxidative damage (Jeng, Loniewska & Wells, 2013; Lotharius & Brundin, 2002; Reiter, 1995), of which as described in Section 1.1.2 Sentinel for defence against oxidative damage are highly dependent on the NADPH supplied by the PPP.

Neurons are capable of using of lactate as an alternative oxidative substrate for ATP generation, allowing the glycolytic pathway to be circumvented and sparing glucose for the PPP (Belanger, Allaman & Magistretti, 2011; Bolanos, Almeida & Moncada, 2010; Itoh et al., 2003; Tsacopoulos & Magistretti, 1996). This diversion of glucose into the PPP plays a crucial role in the regeneration of reduced glutathione (GSH) to combat the accumulation of free-radical mediated damage in neuronal cells (Bolanos, Almeida & Moncada, 2010; Garcia-Nogales, Almeida & Bolanos, 2003; Herrero-Mendez, Almeida, Fernandez, Maestre, Moncada & Bolanos, 2009), which is also required for NADPH production in glial cells such as oligodendrocytes to generate cholesterol-rich myelin sheaths (Kleinridders et al., 2018; Lovatt et al., 2007). Astrocytes, a subtype of glial cells which provide trophic support to neurons, are known to metabolize glucose mainly via glycolysis for the generation of ATP (Kleinridders et al., 2018). This cell type however exhibits higher basal PPP rate compared to neurons (Garcia-Nogales, Almeida & Bolanos, 2003), believed to contribute to the protection of neuronal cells from oxidative damage through the export of GSH, of which neurons have decreased synthesis and concentration of the antioxidant molecule compared to the astrocytes (Makar, Nedergaard, Preuss, Gelbard, Perumal & Cooper, 1994).

An age-dependent decrease in basal NADPH occurs in neurons coupled with a decline in GSH levels (Parihar, Kunz & Brewer, 2008), with impaired GSH regeneration being associated with the development of neurodegenerative diseases such as Alzheimer’s and Parkinson’s disease (Currais & Maher, 2013). Mice genetically modified to be deficient of G6PD, the rate-limiting enzyme of the PPP also exhibited increased lifetime risk of neuropathological damage and oxidative damage within the brain even following a modest decrease of the enzyme activity (Jeng, Loniewska & Wells, 2013) and transgenic over-expression of the G6PD enzyme in neurons of mice also conferred protection against Parkinson’s disease (Mejias et al., 2006). Patients suffering from Ataxia Telangiectasia (AT), another neurodegenerative disease whereby defects exist in the ataxia telangiectasia mutated (ATM) protein, also exhibit abnormally high levels of ROS and low levels of NADPH-dependent antioxidants (McKinnon, 2004). It is speculated that the ATM protein activates G6PD via the association of the enzyme with Hsp27, thereby stabilising the active dimeric/tetrameric forms of G6PD and thus modulating its activity (Cosentino, Grieco & Costanzo, 2011). It is possible that the phenotype of AT, which includes predisposition to cancer, radiosensitivity, sterility, immune dysfunction and neurodegeneration (McKinnon, 2004) may in part be due to increased ROS from decreased G6PD activity.

The precise mechanisms and pathogenesis of amyotrophic lateral sclerosis (ALS), a progressive neurodegenerative disorder characterized by death of motor neurons in the cortex and spinal cord (Kiernan et al., 2011) are not fully understood with the cause and development of the disease believed to be complex and multi-factorial (Chen, Sayana, Zhang & Le, 2013). Oxidative stress has been implicated in ALS pathogenesis, with patients and animal models of ALS exhibiting increased oxidative stress in CNS tissues (Andrus, Fleck, Gurney & Hall, 1998; Barber, Mead & Shaw, 2006; Bogdanov et al., 2000) and 20% of familial ALS being linked to mutations in the copper-zinc superoxide dismutase gene (SOD1) encoding the major antioxidative enzyme CuZnSOD, giving rise to toxicity and death of neurons from protein misfolding or catalysis of oxidative reactions by mutant CuZnSOD proteins (Pansarasa, Bordoni, Diamanti, Sproviero, Gagliardi & Cereda, 2018; Rosen et al., 1993; Valentine & Hart, 2003). Patients with ALS experience an imbalance in the oxidant-antioxidant system, with a reduction in GSH, GR and catalase activity, and the progression of ALS being coupled with reduced G6PD activity in patient’s erythrocytes (Babu et al., 2008). The relationship between G6PD deficiency and 48 the progression of ALS is further supported with reduced metabolite R5P and G6PD activity suggesting perturbations in the PPP being observed in the spinal cords of hSOD1G93A mice (Tefera, Bartlett, Tran, Hodson & Borges, 2019), a benchmark mice model for ALS (Gurney et al., 1994). The physiological significance of the PPP and its role in the development and progression of neurological degeneration suggest that perturbations in G6PD activity may constitute an important risk factor to neurodegenerative diseases, and that treating G6PD deficiency may be a potential therapeutic target for neurodegenerative diseases.

1.4.4 Cardiomyopathies and cardiovascular diseases

Many studies have proposed a link between G6PD deficiency and human cardiovascular disease, with several studies indicating G6PD deficiency having a protective effect against cardiovascular-associated mortality and coronary artery disease (Cocco, Todde, Fornera, Manca, Manca & Sias, 1998; Meloni et al., 2008) and a recent study suggesting an inverse relationship with G6PD-deficient individuals having a greater risk of developing cardiovascular disease (Thomas, Kang, Wyatt, Kim, Mangelsdorff & Weigel, 2018). A study conducted in 1967 also found conflicting observations when cardiovascular health were assessed, whereby G6PD-deficient patients were prone to increased incidence of hypertension and idiopathic cardiomyopathy compared to the background population, but with decreased frequency of coronary artery disease in the affected individuals (Long, Wilson & Frenkel, 1967). Thus, no conclusive relationship between cardiovascular disease and G6PD could be made from observational studies alone.

Contrary to the observation of the possible protective role of G6PD deficiency in population studies, experimental evidence remains unsupportive to the detrimental role of G6PD in human cardiovascular disease. It is well established that cardiomyocytes are highly sensitive to oxidative damage (Lebovitz et al., 1996; Li et al., 1995; Shao, Oka, Brady, Haendeler, Eaton & Sadoshima, 2012) with reliance on G6PD in the maintenance of the cellular antioxidant system and regulation of the cytosolic redox homeostasis in response to oxidative stress (Jain et al., 2003; Jain et al., 2004). G6PD activity increases in response to oxidative injury in myocardial tissue as an antioxidative defence mechanism and to minimize free radical damage to the heart (Jain et al., 2003; Jain et al., 2004; Zimmer, Bünger, Koschine & Steinkopff, 1981), with G6PD-deficient mice exhibiting greater sensitivity to myocardial dysfunction accompanied with the depletion of intracellular thiols and imbalanced redox homeostasis (Hecker, Leopold, Gupte, Recchia & Stanley, 2013; Hecker et al., 2013; Jain et al., 2004). Benfotiamine also decreased oxidative stress and infarct size following myocardial infarction alongside improved survival and overall heart functional parameters (Katare, Caporali, Emanueli & Madeddu, 2010), with the benefit found to be G6PD dependent. Therefore, the G6PD enzyme plays a crucial role in the maintenance of cardiovascular health and may be a possible therapeutic target in the treatment of cardiovascular diseases.

1.4.5 Ageing: healthspan and lifespan

Although many studies have shown G6PD and PPP to be routinely elevated during periods of oxidative stress (Cramer, Cooke, Ginsberg, Kletzien, Stapleton & Ulrich, 1995; Jain et al., 2003; Ursini, Parrella, Rosa, Salzano & Martini, 1997), G6PD activity decreases with age in various cell types such as erythrocytes, lens cells, fibroblasts and in intestinal epithelial cells (Biagiotti, Malatesta, Capellacci, Fattoretti, Gazzanelli & Ninfali, 2002; Duncan, Dell'orco & Guthrie, 1977; Magnani, Stocchi, Bossu, Dacha & Fornaini, 1979), despite an increase in oxidative stress and accumulation with age (Ames, Shigenaga & Hagen, 1993; Noy, Schwartz & Gafni, 1985; Reiter, 1995; Sohal, Mockett & Orr, 2002; Stadtman, 2006). As described in Section 1.1.2 Sentinel for defence against oxidative stress, the PPP is crucial in the prevention of damage from harmful derivatives of oxygen by promoting antioxidative defence in the form of NADPH. This relationship suggests a correlation between G6PD and ageing, with the decrease in G6PD activity potentially resulting in an increase in oxidative stress and the accompanying age- related diseases such as cancer, cardiovascular disease, the decline of the immune system and brain degeneration (Ames, Shigenaga & Hagen, 1993).

Consistent with this hypothesis, over-expression of G6PD has been shown to extend lifespan of the model organism Drosophila melanogaster, with transgenic flies showing enhanced reductive capacity due to the over-expression of G6PD, increased enzymatic activity, NADPH and NADH levels, and increased GSH/GSSG ratio (Legan et al., 2008). Furthermore, G6PD over-expression results in increased resistance against a ROS challenge (Legan et al., 2008; Salvemini, Franze, Iervolino, Filosa, Salzano & Ursini, 1999), without any effect on fertility and metabolic rate of the animal (Legan et al., 2008), and was useful in preventing oxidative stress-induced apoptosis in a number of cell types (Banki, Hutter, Colombo, Gonchoroff & Perl, 1996; Kuo & Tang, 1998; Tian et al., 1998), owing to increased NADPH and glutathione stores to prevent oxidative injury (Leopold, Zhang, Scribner, Stanton & Loscalzo, 2003). This result translated to other species, with G6PD transgenic mice also exhibiting protection against ROS challenges, improved late-life health, and increased overall lifespan in female animals (Nobrega-Pereira et al., 2016).

These observations contribute to the Harman theory (Harman, 1956), or oxidative stress hypothesis of ageing suggesting increased G6PD activity is beneficial, although this theory of oxidative stress as a cause of ageing has been strongly challenged (Gladyshev, 2014; Perez et al., 2009; Viña, Borras, Abdelaziz, Garcia-Valles & Gomez- Cabrera, 2013). G6PD is present in all cells, but is not uniformly expressed across tissues (Battistuzzi, D'Urso, Toniolo, Persico & Luzzatto, 1985), with basal activity varying up to 10-fold amongst different tissues (Corcoran, Fraser, Martini, Luzzatto & Mason, 1996; Kletzien, Harris & Foellmi, 1994). Therefore, effects seen during G6PD over-expression do not always result in the same outcome in all tissues, with studies showing elevation in blood G6PD activity resulting in an increase in NADPH and NADPH oxidase in cardiac muscles, leading to increased ROS production and subsequent cell damage and cell death (Gupte et al., 2009; Serpillon et al., 2009). Over-expression of G6PD is also associated with many age-related diseases such as cancer ultimately resulting in mortality, which was elaborated in Section 1.4.2 G6PD expression in cancer.

G6PD deficiency is the most common human enzymopathy (Beutler & Vulliamy, 2002), with several mutations resulting in severe enzyme deficiency having been discovered using precise molecular characterization (Beutler & Vulliamy, 2002), enabling the opportunity to observe the effect of reduced enzyme activity on human survival. An investigation performed by Petrakis et. al in 1970 on 1,413 African American male individuals in the San Francisco Bay area revealed a lowered incidence of G6PD deficiency in the older aged males when compared to the younger aged counterparts, suggesting accelerated mortality with G6PD deficiency (Petrakis, Wiesenfeld, Sams, Collen, Cutler & Siegelaub, 1970). In contrast, Heller et al. reported a lack of association between pathological adverse effects and overall mortality with G6PD deficiency (Heller, Best, Nelson & Becktel, 1979). The human G6PD variant Hektoen, which is caused by a single amino acid change from histidine to tyrosine, is the only reported G6PD variant at which enzyme activity is above that of normal levels (Dern, McCurdy & Yoshida, 1969), with the increase in enzyme activity being associated with overproduction of the enzyme with no noted changes in enzyme specific activity or structure (Yoshida, 1970). Unfortunately, there were no other research conducted into the longevity or health of patients over-expressing G6PD. As human longevity is multifactorial and highly polygenic, no definite conclusions can be made from observations from these studies. 52

1.5 Conventional therapies targeting the Pentose phosphate pathway

As described in Section 1.4 The Pentose Phosphate Pathway in Ageing and pathophysiology, deviations of PPP activity is associated with numerous pathologies, including diabetes, cancer and ageing. This association has led to research and development for interventions which modulates the PPP for the treatment or management of diseases. In this section, the relevance and use of current pharmacological interventions which alters the PPP via the rate-limiting G6PD enzyme will be discussed, with interventions with substantial evidence being elaborated and this section focusing on both inhibitors and activators of the G6PD enzyme.

1.5.1 G6PD inhibiting agents

The use of pharmacological interventions to disrupt G6PD activity has been proposed to be useful in the management of several diseases such as cancer, diabetes, malaria and other associated disorders involving abnormally elevated G6PD activity which will be detailed in this section. Inhibition of G6PD results in impaired NADPH availability and disruption in the regeneration of reduced glutathione significantly increases the vulnerability of affected cells to apoptosis, which is useful in the treatment of cells whereby there are high demands for both metabolites such as proliferating cancer cells (Jones & Schulze, 2012; Kowalik, Columbano & Perra, 2017; Li et al., 2009) as well as parasite-infected erythrocytes (Zuluaga, Parra, Garrido, Lopez-Munoz, Maya & Blair, 2013). Current chemotherapeutic agents used in the treatment of cancer such as fluoropyrimidine 5-fluorouracil and Adriamycin induce tumour cell death by eliciting ROS damage as part of their mechanism of action (Longley, Harkin & Johnston, 2003). As the PPP plays a crucial role in the generation of reducing power in the form of NADPH, necessary for eliciting antioxidative defence against ROS (described in Section 1.1.2 Sentinel for defence against oxidative stress), inhibition of the PPP by its rate- limiting enzyme G6PD may enhance the sensitivity of cancer cells to the effect of chemotherapy via combinational treatment with G6PD inhibitors (Jones & Schulze, 2012; Li, Fath, Scarbrough, Watson & Spitz, 2015).

A steady source of NADPH is also essential for the survival of parasites as it is involved in nucleotide synthesis as well as other biosynthetic pathways through the supply of carbohydrate intermediates (Muller, 2004), and thus inhibition of the PPP is suitable as a target for parasitic diseases. PPP activation may also result in the exacerbation of pro-oxidant and pro-inflammatory pathways in the diseased state through NADPH oxidase activation, with reduction of NAPDH oxidase activity or PPP blockade in diabetic animals resulting in the prevention of tissue dysfunctions (Gok et al., 2016; Peiro et al., 2016). The use of G6PD inhibitors may also prove beneficial in the management of obesity, as haematopoietic G6PD had also been suggested as a pharmacological target for the reduction of pro-inflammatory factors affecting adipose tissue and consequently whole body energy homeostasis in the context of obesity (Park, Choe, Sohn & Kim, 2017).

Current known inhibitors of G6PD that have been brought forward as potential therapeutics includes the compounds 6-aminonicotinamide, glucosamine, catechin gallates and dehydroepiandrosterone (DHEA), with active research pursuing new inhibitors of G6PD in recent years (Ghashghaeinia et al., 2016; Mele et al., 2018; Preuss et al., 2013; Shin et al., 2008). Administration of the steroid hormone DHEA which inhibits the G6PD enzyme in an uncompetitive manner (Marks & Banks, 1960) prevents tumour progression and proliferation (Lopez-Marure, Contreras & Dillon, 2011; Simile et al., 1995), with G6PD inhibition also restoring the sensitivity of cancer cells to chemotherapy (Jones & Schulze, 2012; Li, Fath, Scarbrough, Watson & Spitz, 2015). 6- aminonicotinamide is a specific inhibitor of the PPP, acting competitively on NADP+- dependent enzymes of the pathway (De Preter et al., 2015; Köhler, Barrach & Neubert, 1970; Koutcher, Alfieri, Matei, Meyer, Street & Martin, 1996) and glucosamine inhibits G6PD in a nonhormonal, competitive manner once converted to GlcN-6-phosphate during transport into cells (Glaser & Brown, 1955; Kanji, Toews & Carper, 1976). Gallated catechins are potent inhibitors of G6PD and other enzymes which employ NADP+ as coenzymes, such as 6PGD, isocitrate dehydrogenase, fatty acid synthase and glutamate dehydrogenase (Shin et al., 2008), making the direct effect of catechins inhibition on the G6PD enzyme unclear.

There are also other proposed pharmacological treatments which alter PPP activity through indirect modulation of G6PD enzyme expression. These includes Zoledronic acid (ZA), which is a standard therapy for the management of bone metastasis and osteoporosis, reducing cell proliferation and the PPP via Ras signalling inhibition with reduced expression of the G6PD enzyme observed resulting in the reduction of bladder cancer cell proliferation (Wang et al., 2015). Resveratrol, found in the skin of red grapes and peanuts (Jang et al., 1997) also downregulated key enzymes in the PPP G6PD and TKT leading to suppressed cell cycle progression (Vanamala, Radhakrishnan, Reddivari, Bhat & Ptitsyn, 2011), with many other agents having been suggested as inhibitors of G6PD such as CB83 (Preuss et al., 2013). Polydatin, a glucoside of resveratrol has also recently been reported to directly inhibit G6PD in a dose-dependent manner resulting in a raised NADP+/NADPH ratio and redox imbalance, although the mechanism of action and interaction to the G6PD protein remains to be elucidated (Mele et al., 2018).

Although the inhibitors DHEA and 6-aminonicotinamide mentioned above are well-studied and characterized, there still exists a need for new G6PD inhibitors due to the limitations of the current known inhibitors of G6PD. 6-aminonicotinamide is known to have toxic side effects such as nerve damage and vitamin B deficiency (Gupte, 2008), whilst DHEA is a naturally occurring hormone that is known to have pleiotropic effects such as growth inhibition that is not related to the inhibition of G6PD (Biaglow, Ayene, Koch, Donahue, Stamato & Tuttle, 2000; Ng, Wang, Lee & Hu, 1999; Paulin, Meloche, Jacob, Bisserier, Courboulin & Bonnet, 2011), and treatment with DHEA can have detrimental side effects such as the induction of autoinflammatory reactions (Nyce, 2017). The efficacy of DHEA as a G6PD inhibitor in vivo is disputable with the drug being converted into other hormones and only inhibiting the enzyme at high concentrations in vivo (Di Monaco et al., 1997). Furthermore, the antiproliferative effects of DHEA on cancer cells are most likely due to mitochondrial impairment leading to cessation of cell growth rather than G6PD inhibition (Ho, Cheng, Chiu, Weng & Chiu, 2008). Due to the lack of specificity for these inhibitors, there may be a need for the identification of other inhibitors of G6PD with higher specificity, potency and with fewer side effects as potential pharmacological treatment for diseases requiring high consumption for NADPH and GSH such as malaria and cancer.

1.5.2 G6PD activating agents

There are no pharmacological agents for the treatment of G6PD deficiency, with management mainly consisting of avoidance of the triggering agents accompanied with supportive care to alleviate the symptoms of the deficiency, all of which have their limitations as described in Section 1.3.3 Management of G6PD deficiency. A recent study conducted by Hwang et al. identified a possible novel small molecule agent which activates and stabilises the G6PD enzyme to manage the challenges associated with the deficiency (Hwang et al., 2018). The small molecule 2,2’-disulfanediylbis(N-(2-(1H- indol3-yl)ethyl)ethan-1-amine), termed AG1 improves G6PD oligomerization by bridging the dimer interface at the NADP+ substrate binding sites of interacting G6PD monomers, ultimately stabilising the enzyme and preventing activity loss (Hwang et al., 2018; Raub et al., 2019). Trials of the small molecule on recombinant G6PD enzyme showed AG1 increased wild-type G6PD enzyme activity and restored the activity of several enzyme deficiency variants, with changes to enzyme kinetics being observed in the G6PD_Canton suggesting possible facilitation of improved substrate binding to the enzyme. These observations were coupled with reduced susceptibility of the AG1-treated enzyme to proteolysis, and improved cell viability with reduction in ROS levels following AG1 treatment (Hwang et al., 2018). Furthermore, AG1 reduces haemolysis in stress- induced erythrocytes (Hwang et al., 2018), which may be effective in the management of G6PD deficiency of which sporadic haemolysis is a common symptom as elaborated in Section 1.3.2 Phenotype and clinical manifestations.

This study is not dissimilar to the main aim of this project, which was to identify novel small molecules which increases overall G6PD enzyme activity, with focus on lifespan and healthspan improvement rather than just the management of G6PD deficiency. As described in Section 1.4.5 Ageing: healthspan and lifespan, there are some evidence of beneficial effects on longevity with elevation of G6PD activity, with animals over-expressing G6PD having better survival and lifespan (Legan et al., 2008; Nobrega-Pereira et al., 2016). No conclusive relationship between G6PD deficiency in humans and longevity could be made from previous observational studies (Heller, Best, Nelson & Becktel, 1979; Petrakis, Wiesenfeld, Sams, Collen, Cutler & Siegelaub, 1970), with conflicting evidence having been reported likely due to the highly polygenic and multifactorial nature affecting of longevity.

Although there are few known agents which directly increases G6PD enzyme activity, with the G6PD-activating small molecule AG1 only recently being identified by Hwang et al., several treatments are known to indirectly increase PPP shunt.

Benfotiamine (BFT), a synthetic S-acyl derivative of thiamine (Vitamin B1) is a transketolase activator which prevents the progression of diabetic complications such as neuropathy, nephropathy and retinopathy by directing precursors of advanced glycation end products into the PPP, increasing the pathway shunt and raising G6PD activity indirectly (Balakumar, Rohilla, Krishan, Solairaj & Thangathirupathi, 2010). BFT however has not been trialled in the context of G6PD deficiency but has been extensively researched for use in the management of diabetes and non-diabetic-associated pathologies (Balakumar, Rohilla, Krishan, Solairaj & Thangathirupathi, 2010; Katare, Caporali, Emanueli & Madeddu, 2010; Katare et al., 2013; Stracke et al., 2001). The lack of G6PD activating agents and the potential benefits of PPP elevation highlights the need for the identification for novel G6PD activating pharmacological drugs.

Chapter 2 High- throughput screen for small molecule G6PD activators

High-throughput screen for small molecule G6PD activators

2.1 Introduction

Methods for the measurement of G6PD enzyme activity from physiological samples are well described, with both direct and indirect modes of detection being commonplace. Direct assays includes the quantitative measurement of gross G6PD activity from lysates (Glock & McLean, 1952), directly measuring the production of G6PD enzyme product NADPH which absorbs light at 340 nm and fluoresces at 450 nm, and the qualitative equivalent of this assay is the fluorescent spot test (Beutler & Halasz, 1966), which assesses the presence of fluorescence in a sample following incubation with the enzyme substrates. G6PD activity can also be measured indirectly, via the use of chromophores such as brilliant cresyl blue (Dickens & Glock, 1951), resazurin (Guilbault & Kramer, 1965) or formazan derivatives (Jalloh et al., 2004), which uses NADPH availability to produce a colorimetric change.

There were several factors which dictated which assay type we could use for a high-throughput screen (HTS) to identify compounds which activate the enzyme G6PD. As we plan to assess the effect of drugs on the activity of purified recombinant wild-type G6PD enzymes, we decided to proceed with optimizing and developing a direct, absorbance-based assay, measuring the enzymatic production of NADPH which has an absorbance at a wavelength of 340 nm (Ziegenhorn, Senn & Bucher, 1976). In this chapter, the detection of G6PD enzyme activity was optimized in order to be compatible with a high-throughput screening protocol. The parameters for high-throughput screens are generally optimized for each assay, due to the diversity of enzyme features which dictate assay conditions. Variables such as optimum enzymatic substrate concentrations per reaction, template layout, temperature, appropriate controls, method to terminate enzyme activity and assay readouts had to be each optimized for our screen.

The optimum concentrations for G6PD substrates NADP+ and G6P to be used in the screen were determined via assessing the G6PD enzyme kinetics, which are batch specific, and necessary to determine optimum substrate concentration such that the substrates do not saturate the enzyme nor is insufficient to engage and promote enzyme activity. A suitable assay stop solution is also required, which not only effectively halts enzyme activity at the time of administration, but also does not affect the read out of the assay, which will be an absorbance readout at 340nm. Furthermore, the amount of enzyme 59 required for each reaction must also be determined, with enough enzyme needed to obtain activity, but not too much that will deplete the substrates too quickly for enzyme activity to be compared.

For this project, compounds were randomly selected from the WECC (WEHI- CCIA) diversity library generated by the Walter and Eliza Hall Institute (WEHI) and the Children’s Cancer Institute Australia (CCIA) to assess for G6PD activating capabilities, with the drugs being dissolved and dispensed in DMSO to a final reaction concentration of 10 µM. The recombinant G6PD enzyme to be used in the screen will have to be assessed to determine if the presence of DMSO in the reaction will impact on the activity of the G6PD enzyme. Thus, this chapter focuses on the developing, optimization, validation and finally the execution of the G6PD activity HTS which will screen 10,240 novel small molecules to identify drugs which activates the G6PD enzyme. These drugs will then be further characterised in Chapter 3.

2.2 Materials

2.2.1 Materials and reagents

Materials

Compound library

The compounds screened in this study were from the WECC (WEHI-CCIA) diversity library; 10,240 drug-like molecules adapted from four independent vendors, filtered for reactive functional groups and pan-assay interfering compounds (PAINS) (Dahlin et al., 2015). The library is also optimized to consider compounds of 85% similarity to any other compounds as determined by the Tanimoto coefficient. The compounds were screened at the concentration of 10 µM, with DMSO concentration kept constant at 0.2%.

Reagents

G6PD Screen start solution [50 mM Tris-HCl, pH 7.4, 1 mM G6P]

G6PD Screen Substrate-enzyme mix [50 mM Tris-HCl, pH 7.4, 1.6 mM NADP+, 2.5 µg/ml recombinant human wild-type G6PD enzyme]

G6PD Screen stop solution [6 M GdnHCl]

G6PD Screen saturation mix [50 mM Tris-HCl pH 7.4, 10 mM G6P, 2.4 mM NADP+]

G6PD Screen blank mix [50 mM Tris-HCl, pH 7.4]

2.2.2 Instruments

SpectraMax M Multi-mode microplate reader (Molecular Devices), Thermo Multidrop 384 and Multidrop Combi (Thermofisher Scientific, Hudson, NH), EnSpire® EnVision Multimode Plate Reader (PerkinElmer).

2.3 Experimental Methods

2.3.1 G6PD kinetic studies

The Km and Vmax of G6PD enzyme were determined using Lineweaver-Burk curves (Lineweaver & Burk, 1934). These were obtained for by titrating NADP+ (0 – 1000 µM) while keeping constant saturating concentration of G6P. This was performed similarly for G6P with a fixed saturating concentration of NADP+ and varying G6P concentrations (0 – 8000 µM). All kinetic studies were performed at 24 ºC and at optimal pH (50 mM Tris-HCl, pH 7.4), with absorbance of the reaction measured at 340 nm every

20 seconds, and the initial slope, Km and Vmax values were calculated by nonlinear regression using Graphpad Prism 7 software (Graphpad, San Diego, CA).

2.3.2 HTS screen stop solution optimization

Varying concentrations of GdnHCl (0 - 2 M) were trialled for efficacy in terminating G6PD enzyme activity and maintaining NADPH absorbance for measurement. Standard G6PD enzyme assay conditions were used, with each reaction mixture consisting of 50 mM Tris-HCl, pH 7.4, 0.3 mM NADP+, 1.5 µg enzyme and 0.5 mM G6P, and the reaction being initiated with G6P addition. NADPH production was measured by reading absorbance at 340 nm every 20 seconds. After 12 minutes of reaction, varying concentrations of GdnHCl were added to terminate the reaction, before reading the absorbance after stop solution addition until 40 minutes of reaction has occurred. The appropriate stop solution concentration was determined based on the complete cessation of enzyme activity with the addition of the solution, and the maintenance of absorbance at 340 nm after stop solution addition.

2.3.3 G6PD enzyme titration for HTS screen

G6PD activity was determined as described previously (Tian et al., 1998), with modifications. Each reaction mixture consists of 50 mM Tris-HCl (pH 7.4), 0.3 mM NADP+, varying amounts of G6PD enzyme per reaction (0 – 0.5 µg enzyme) and with the presence of drug or vehicle control. Reactions were initiated with the addition of substrate G6P to a final reaction concentration of 0.5 mM and activity was measured using a spectrophotometer at room temperature (24 °C). NADPH production was measured at 340 nm every 20 seconds for at least 20 minutes, and the rate of NADPH production determined as a unit of AU/hour from the initial 2 – 5 minutes of the initial slope. The appropriate enzyme concentration was chosen based on reaction rate linearity. Enzyme titration was performed on the same protein batch used as the HTS G6PD screen.

2.3.4 G6PD enzyme DMSO tolerance test

The G6PD enzyme activity assay was trialled using varying DMSO concentrations from 0% to 1%, under standard G6PD enzyme activity conditions (Experimental methods section 2.3.3) with 0.5 µg enzyme per reaction. The linear reaction was obtained utilizing the first 10 minutes of the reaction, and the reaction rates were plotted as average and standard deviation of three replicates against DMSO concentration using Graphpad Prism software.

2.3.5 HTS G6PD enzyme activator screen

Compounds were derived from the WECC-CCIA library (CCIA, UNSW Sydney), and were administered into 384- well plates (PerkinElmer: 6007649) for small volume by the screening compound facility. Screening plates were stored in -20 ºC upon arrival, and the plates were centrifuged prior to use. For the controls, 20 µl of the G6PD screen start solution (final assay concentration 50 mM Tris, 0.5 mM G6P) were added to wells A24 – H24, and 20 µl of the G6PD screen saturation mix (final assay concentration 50 mM Tris, 2.0 mM NADP+, 5 mM G6P) to wells I24 – P24 at the beginning of the screen. 20 ul of the G6PD screen enzyme-substrate mix (final assay concentration 50 mM Tris-HCl, 0.8 mM NADP+, 0.05 µg G6PD enzyme) were added to all wells using the Thermo Multidrop Combi reagent dispenser (Thermofisher Scientific, Hudson, NH) and the drug- enzyme mixture allowed to equilibrate for 15 minutes. The reactions were then initiated with the addition of 20 µl of G6PD screen start solution (final assay concentration 50 mM Tris, 0.5 mM G6P) to columns 2 - 23. Column 1 serves as a blank well with 20 µl G6PD screen blank mix addition. After 5 minutes of reaction, all enzyme activity were halted with the addition of 20 µl G6PD screen stop solution (final assay concentration 2.0 M GdnHCl) to all wells. The plates were then centrifuged at 1000 g for 1 minute, and endpoint absorbance read at 340 nm. Analyses were conducted using EnSpire Workstation software version 4.10.3005.1440, with enzyme activity determined by comparing endpoint absorbance produced between treatment groups to the solvent control.

2.3.6 G6PD enzyme activator/inhibitor HTS hits validation screen

Several compounds were cherry-picked for a secondary screen, with a large proportion of compounds showing >15% activation of the G6PD enzyme and a fraction showing >15% inhibition of the G6PD enzyme being subjected to the secondary screen. The compounds were cherry-picked following analyses using the Enspire Workstation software version 4.10.3005.1440 and were administered into 384- well plates (PerkinElmer: 6007649) by the screening compound facility. Screening plates were stored in -20 ºC upon arrival and thawed and centrifuged prior to use. The assay was performed as described before (Experimental methods section 2.3.5) with each compound tested in triplicate under the same conditions, controls and using the same protein batch. In brief, 20 µl enzyme-substrate mix were added to the wells containing the compounds and allowed to equilibrate for 15 minutes. The reactions were initiated with the addition of 20 µl G6PD screen start solution, and the reactions occurring for 5 minutes. The reactions were terminated with the addition of 20 µl G6PD screen stop solution, and the plates centrifuged, and absorbance read at 340 nm. Analyses were conducted using EnSpire Workstation software version 4.10.3005.1440, with enzyme activity determined by comparing endpoint absorbance produced between treatment groups to the solvent control.

2.3.7 G6PD activator/inhibitor Hits EC50/IC50 determination

The top compound hits were manually cherry-picked and were subjected to a tertiary log dose validation screen to determine the activator/inhibitor EC50/IC50. The assay was performed as described before (Experimental methods section 2.3.5) with the addition of varying the compound concentrations from 0 – 20 µM, with each compound tested in triplicate, under the same conditions and using the same protein batch. Analyses were performed using EnSpire Workstation software version 4.10.3005.1440, with drug concentration at which half-maximal response was achieved, EC50/IC50 were determined from the graphs. In brief, 20 µl enzyme-substrate mix were added to the wells containing the compounds and allowed to equilibrate for 15 minutes. The reactions were initiated with the addition of 20 µl G6PD screen start solution, and the reactions were allowed to proceed for 5 minutes. The reactions were terminated with the addition of 20 µl G6PD screen stop solution, and the plates centrifuged before absorbance read at 340 nm. Enzyme activity were represented by the endpoint absorbance, with comparison to solvent control representing 100% enzyme activity.

2.4 Results

2.4.1 G6PD enzyme assay optimization for HTS

Optimization of G6PD amount for enzyme activity assay

The G6PD HTS will screen 10,240 small molecules with drug-like properties against recombinant wild-type human G6PD enzyme to identify candidates that increase overall activity of the enzyme. Here, we aimed to determine the appropriate quantity of the enzyme required for each reaction, generating enough activity to measure and compare differences in the rate of NADPH production within an allocated timeframe, whilst not being too excessive such that the substrates present in the reaction are exhausted before a difference in reaction rate can be determined. Furthermore, the development of enzymatic assays recommend having a general rule of intensity of signal exceeding at least two fold the baseline of the assay, that is, having a signal to background ratio (S/B) of at least 2 (Bisswanger, 2014).

Increasing amounts of G6PD enzyme of up to 0.5 µg per reaction were trialled in a preliminary G6PD enzyme activity assay to determine the optimum enzyme amount necessary to generate a linear rate of NADPH production within 20 minutes. The maximum absorbance, or total assay absorbance, was defined as the complete conversion of NADP+ to NADPH, that is, when the NADP+ is exhausted producing absorbance reaches a maximum saturation of 0.663 AU. Reactions containing 0.001 µg and 0.005 µg enzyme had an activity rate of 0.009 AU/hour and 0.088 AU/hour respectively, with the reactions producing a mean absorbance of 0.006 AU and 0.029 AU at after 20 minutes of reaction respectively (Figure 2.4.1.1). As these values are only a small fraction of the total absorbance produced when all NADP+ were converted, and do not abide by the appropriate S/B, the amount of NADPH produced by 0.001 µg and 0.005 µg enzyme were determined to be insufficient to detect variations in NADPH production, and the rate of production could be increased to reach that of the saturation by increasing enzyme amount.

G 6 P D e n z y m e a c tiv ity a ss a y w ith v a r y in g a m o u n ts o f r e c o m b in a n t G 6 P D e n z y m e

0 .8

C o n tro l

0 .6 0 .0 0 1 µ g G 6 P D _ W T 0 .0 0 5 µ g G 6 P D _ W T

e 0 .0 1 µ g G 6 P D _ W T )

g 0 .4 n

m 0 .0 2 5 µ g G 6 P D _ W T

0 0 .0 5 µ g G 6 P D _ W T

3 U

( 0 .2 0 .1 µ g G 6 P D _ W T A 0 .5 µ g G 6 P D _ W T

0 .0 5 1 0 1 5 2 0 2 5 T im e (m in u te s ) -0 .2

Figure 2.4.1.1 Optimization of G6PD concentration for high-throughput assay development. Varying quantities of recombinant human wild-type G6PD enzymes of up to 0.5 µg were tested to determine the optimum NADPH production rate required to measure G6PD activity. NADPH production rates were linear for amounts of G6PD enzyme of 0.05 µg per reaction and below, with 0.05 µg enzyme per reaction having a mean NADPH production rate of 1.401 AU/hour. 0.1 µg enzyme per reaction resulted in plateauing NADPH production towards the end of the reaction time, with a linear NADPH production rate of 3.052 AU/hour AU/hour and 0.5 µg enzyme group reaching saturation within 10 minutes reaction. Each enzyme amount group were tested in triplicate and represented as mean value with standard deviation.

NADPH production rates were also linear in reactions containing 0.01 µg and 0.025 µg enzyme, with an activity rate of 0.187 AU/hour and 0.582 AU/hour produced respectively. The 0.01 µg enzyme reaction produced a total of 0.063 AU after 20 minutes of reaction, a 9.5% fraction of the total absorbance maximum which is too low for enzyme activity comparison. Total absorbance produced by 0.025 µg was 0.175 AU, 26.4% fraction of the maximum absorbance of the assay (Figure 2.4.1.1). This NADPH production rate was enough to detect activators of the enzyme, which would cause an increase in NADPH production, while having an appropriate absorbance gap of about 74% to accommodate for the increase in absorbance before saturation is reached. Similarly, NADPH production by 0.05 µg enzyme resulted in a linear reaction rate of 1.401 AU/hour with a maximum of 0.369 AU produced from the reaction. This is a 56% fraction of the total absorbance of the assay, which allowed for deviations in NADPH production to be identified, whilst allowing for changes in the total absorbance produced to occur.

Reactions containing 0.1 µg enzyme had an initial reaction rate of 3.052 AU/hour in the first 10 minutes of the reaction, with enzyme activity decreasing as the reaction progressed, and the final total absorbance of 0.592 AU being produced from the reaction. This is a fraction of 89.3% of the total assay absorbance, with only 10.7% leeway for increases in absorbance to occur prior to reaching assay saturation. Reactions occurring with this NADPH production rate would be able to show deviations in enzyme activity if the reaction time is kept within the 10 minutes reaction range, whereby the reaction rate is linear. 0.5 µg enzyme produces 10.33 AU/hour, with the absorbance reaching saturation within 10 minutes of reaction initiation. From these results, enzyme amounts of 0.05 µg per reaction produced the optimum activity rate, reaching a maximum absorbance that falls in between the absorbance saturation and absorbance baseline. Thus, the HTS for the identification of G6PD activating small molecules will be conducted within reactions containing 0.05 µg enzyme.

Determining substrate concentrations for G6PD HTS

The substrate concentration for the G6PD enzyme activity assay should ideally be + close to the Km for the G6PD enzyme substrates NADP and G6P, to achieve half the maximum rate of product NADPH production by the enzyme. The enzyme kinetics of the protein batch used in the G6PD HTS were therefore determined. Enzyme kinetics of the G6PD protein were determined by varying the concentrations of the enzyme substrates NADP+ and G6P as described in Experimental methods section 2.3.1, to obtain Michaelis-Menten kinetics of the enzyme.

The Km and Vmax values were determined using the Michaelis-Menten equation, with each reaction performed with three technical replicates. Utilizing the Michaelis-

Menten curve, the Km of G6PD substrate was determined to be 0.196 ± 0.0215 mM, with the Vmax being 5.712 ± 0.1262 reaction velocity units (Figure 2.4.1.2.A). These values suggest the optimum working concentration for the G6PD enzyme activity assay to be at 0.196 mM to achieve half the rate of NADPH production, allowing any deviations to the enzyme activity caused by the addition of the small molecule drugs to be detected. The + Km for substrate NADP was much lower than that of G6P, with the Km being determined to be 0.02127 ± 0.0033 mM, achieving a Vmax of 6.042 ± 0.1058 reaction velocity unit at a much lower substrate concentration (Figure 2.4.1.2.B).

Figure 2.4.1.2 Km determination for G6PD substrates for high-throughput screening optimization.

(A) Michaelis-Menten kinetics for Km of substrate G6P. Km of substrate G6P for recombinant wild-type

G6PD enzyme was determined to be 0.196 ± 0.0215 mM, with Vmax of 5.712 ± 0.1262 reaction velocity. + + (R square = 0.9299). (B) Michaelis-Menten kinetics for Km of substrate NADP . Km of substrate NADP was determined to be 0.02127 ± 0.0033 mM with Vmax of 6.042 ± 0.1058 reaction velocity (R square = 0.9402). (C) Lineweaver-Burk plots for substrate G6P and (D) for substrate NADP+.

Lineweaver-Burk plots were also graphed utilising the varying substrate concentrations of each respective substrates of the G6PD enzyme and the reaction velocity of the enzyme at each substrate concentration. The Lineweaver-Burk plot makes the values of the x-intercept and y-intercept known through extrapolation of the line of 1 1 best fit, with x-intercept = - and y-intercept = (Lineweaver & Burk, 1934). From Km Vmax the Lineweaver-Burk graphs, the curve equations are determined as shown:

Equation 5: The Lineweaver-Burk equation for substrate G6P,

y = 0.02371 * x + 0.1921, R square = 0.7704 (Figure 2.4.1.2.C)

Equation 6: The Lineweaver-Burk equation for substrate NADP+,

y = 0.003948 * x + 0.1644, R square = 0.8194 (Figure 2.4.1.2.D)

The x-intercept was determined to be -8.10207 from the G6P Lineweaver-Burk 1 Equation 5. By substituting this value into the equation x-intercept = - , the Km for Km substrate G6P was calculated to be 0.123 mM, which is lower than the value obtained from the Michaelis-Menten equation (Figure 2.4.1.2.A). Vmax was determined by extrapolating the line of best fit on the Lineweaver-Burk plot to obtain the y-intercept and 1 substituting the y-intercept into the formula y-intercept = , with the y-intercept of Vmax

G6P being 0.1921 (Equation 5), giving the Vmax of substrate G6P of 5.206 AU/hour, also lower than the Vmax determined from the Graphpad software. Using Equation 6, the x- intercept for the NADP+ Lineweaver-Burk equation was determined to be -41.6413.

1 + Substituting this value into the equation x-intercept = - results in the Km for NADP Km being calculated to be 0.024 mM, slightly higher than the value determined from the Michaelis-Menten graph analysis (Figure 2.4.1.2.B). The y-intercept determined from extrapolating the line of best fit was determined to be 0.1644, resulting in a Vmax of 1 6.083AU/hour using the formula y-intercept = . Vmax

Assuming there was a 1:1 conversion of NADP+ to NADPH by the G6PD enzyme + within a reaction containing the substrates at Km (0.12 mM G6P and 0.02 mM NADP ), the maximum concentration of NADPH that will result from conversion will be 0.02 mM, or 0.8 nmol in a reaction volume of 40 µl. NADPH is not detectable spectrophotometrically at 340 nm at this concentration (Supplementary 1). Substrate concentrations had to be increased above that of the determined Km values, and we proceeded with substrate concentrations of 0.5 mM for G6P, and 0.8 mM for NADP+.

By substituting the concentration [S] of 0.5 mM G6P into Equation 5, whereby the x axis represents 1/[S], the rate of enzyme reaction was determined to be 4.175 AU/hour when G6P was present at this concentration. This is 73.09% of the maximum velocity as determined from the Michaelis-Menten curve when varying G6P in the presence of saturating NADP+. Similarly, using Equation 6, NADP+ at a concentration of 0.8 mM will generate a reaction velocity of 5.905 AU/hour, 97.74% of the maximum velocity. By assuming complete conversion of NADP+ to NADPH, the amount of NADPH present in the final volume 40 µl at 0.8 mM concentration will be 32 nmol, which will result in an absorbance of 1.421 AU when the conversion of NADP+ to NADPH is at completion, a 12.18-fold of the baseline absorbance of the reaction (Supplementary 1). This large margin provided a wide range for which variations in G6PD activity could be compared and measured. Absorbance produced by NADPH of concentrations above 1.0 mM within the reaction reach saturation and are not suitable for the assay.

The substrate concentration for the G6PD HTS were optimized to final concentrations of 0.5 mM G6P, and 0.8 mM NADP+, which provided an appropriate readout range for variations in G6PD activity to be measured without the absorbance reaching saturation.

Optimising G6PD enzyme activity assay stop solution

In order to terminate enzyme activity after a reaction period, and maintain the absorbance or fluorescence produced by the reaction for measurement which may occur sometime after termination of the enzyme activity, it was necessary to optimize a stop solution for this HTS. This should be fast acting and involve only a single step, in order to be compatible with the large number of assays conducted in a HTS. An appropriate stop solution was needed to terminate and cease recombinant G6PD activity, as well as maintain and prevent any changes to the absorbance present by the produced NADPH until the plates can be measured spectrophotometrically.

Guanidine hydrochloride (GdnHCl), commonly used for protein unfolding (Wingfield, 2001), was trialled at varying concentrations from 0 – 2.0 M concentration as an assay stop solution. A 6.0 M solution of commercial GdnHCl (>99%) is clear and colourless and does not produce any absorbance at the 340 nm wavelength when trialled in a pilot test. GdnHCl at concentrations under 1.0 M did not effectively terminate G6PD enzyme activity, which continued despite the addition of the stop solution. Concentrations of GdnHCl at 1.5 M and 2.0 M sufficiently terminated NADPH production, with the absorbance of existing NADPH in the solution being maintained and stable for up to 27.5 minutes after the stop solution addition (Figure 2.4.1.3). From these results, we determined that 2.0 M GdnHCl was appropriate as a stop solution for the G6PD HTS, with the solution halting any further enzyme activity upon addition into the reaction, and the solution not altering the absorbance produced by the NADPH for approximately 30 minutes, which provides a leeway of time from when the enzyme activity is terminated until the plates are measured.

C o n ce n tr a tio n o f G u a n id in e h y d r o ch lo rid e (G d n H C l) fo r e n z y m a tic a c tiv ity sto p so lu tio n

1 .0 G d n H C l A d d it io n C o n tro l

e 0 .5 M G d n H C l

n m

a 1 .0 M G d n H C l

0 o

4 1 .5 M G d n H C l s

3 0 .5 b

2 .0 M G d n H C l A

0 .0 0 1 0 2 0 3 0 4 0 5 0 T im e (m in u te s )

Figure 2.4.1.3 Optimization of GdnHCl stop solution concentration for G6PD HTS development. Varying concentrations of GdnHCl were assessed for their ability to stop G6PD enzyme activity assay using 1.5 µg recombinant human wild-type G6PD enzyme following 12 minutes of enzymatic reaction. The control reaction of H2O addition at 12 minutes did not halt G6PD activity, with NADPH production continuing up to 40 minutes of reaction time. Concentrations of 1.5 M GdnHCl and above blocked NADPH production, with the absorbance of produced NADPH being maintained up to 27.5 minutes post- stop solution addition. Each reaction was performed in triplicate, represented as mean absorbance with standard deviation.

Assessing G6PD enzyme DMSO tolerance

Molecules randomly selected from the WECC-CCIA diversity library were dispensed into 384- well plates by the drug dispensary instrument, which were then diluted to the desired dose of 10 µM with the addition of the enzyme mixture, substrates and assay start solution. The molecules were dispensed from stock solutions of dimethyl sulfoxide (DMSO) solvent, resulting in DMSO present in the reaction to a fixed final concentration of 0.2% in all reactions. Before initiating the screen, the effect of DMSO on the enzyme activity was determined, as the presence of DMSO in the solution may cause conformational changes to the protein which may result in changes to the activity of the enzyme. DMSO tolerance of the G6PD enzyme was explored by performing enzyme activity assays with titrating DMSO concentrations of up to 1.0%, under standard enzyme activity conditions and at room temperature. The enzyme reactions were allowed to proceed for 5 minutes, and NADPH production by the enzyme were measured at absorbance 340 nm. The rate of reaction was determined for the enzymes in the presence of all DMSO concentrations.

G6PD enzyme activity in the absence of DMSO had a mean NADPH production rate of 3.146 ± 0.072 AU/hour, with no significant difference observed in reactions where the enzymes were incubated in the presence of varying concentrations of DMSO of up to 1.0%. A final reaction DMSO concentration of 0.2% displayed an activity rate of 3.436 ± 0.116 AU/hour, while DMSO concentrations of 0.4%, 0.8% and 1.0% had activity rates of 3.121 ± 0.073 AU/hour, 3.133 ± 0.078 AU/hour and 3.081 ± 0.051 AU/hour respectively. The presence of DMSO at up to a concentration of 1.0% did not significantly alter the NADPH production rate by G6PD enzyme. Therefore, 0.2% DMSO, which will be present in all reactions conducted in the G6PD HTS, would not interfere with enzyme activity and was deemed compatible in the G6PD screen.

G 6 P D E n z y m e D M S O to le r a n c e te st

4 .5

n s n

o 4 .0

)

o r

h 3 .5

(

P D

A 3 .0 N

2 .5 0 0 .2 0 .4 0 .8 1 .0

D M S O (% )

Figure 2.4.1.4 G6PD enzyme DMSO tolerance test for HTS optimization. Recombinant human wild- type G6PD protein to be used in the HTS screen were exposed to varying concentrations of DMSO to determine the effect of the drug solvent on the enzyme. Small molecules from the WECC-CCIA library are administered into the 384- well plates dissolved in the solvent DMSO and will be at a final reaction concentration of 0.2%. The assay was conducted in standard G6PD screen conditions. There were no significant differences in G6PD enzyme activity in the presence of DMSO at up to 1.0% in concentration. Each datapoint represents an individual reaction, with statistical analysis performed using one-way ANOVA, standard error of mean displayed.

2.4.2 G6PD HTS validation

G6PD HTS practical considerations

The G6PD enzyme activity assay was optimized for high-throughput assessment of 10,240 randomly selected small molecules, with the enzyme amount, reaction substrate concentration, stop solution concentration and enzyme DMSO tolerance having been optimized for the screen, to be performed at room temperature for ease of handling. The G6PD enzyme activity screen was optimized for 384- well plates, with drugs having been dispensed into the allocated treatment wells (Figure 2.4.2.1) by the drug dispensary. Each plate had the capacity to test 256 unique small molecules, consuming 40 plates in total for the testing of all compounds selected from the library.

The assay plates in the screen were standardized by implementing appropriate controls within the assay plate layout. Every reaction consisted of 0.05 µg G6PD enzyme and NADP+, which was dispensed using an automated multidrop dispenser at the start of the experiment. Reaction blanks were included on either side of the plate in columns 1, 2, 23 and 24, as a control to ensure there was no variation in the orientation of the plate and to serve as a reaction baseline or signal background. Each plate also included DMSO control reactions in columns 3 and 4, rows A – H, and a negative control group using known G6PD inhibitor DHEA at a concentration of 10 µM in the same columns, rows I – P. The DMSO and DHEA were dispensed by the drug dispensary machine. Reactions with excess substrates, with a final reaction concentration of 5 mM G6P and 2 mM NADP+, were included in the assay layout in columns 21 and 22, rows A – H to control for assay-based absorbance saturation. The substrate concentrations were increased by initiating the reactions with the addition of G6PD screen saturation mix in place of the G6PD screen start solution. Each plate also includes an assay relative saturation group in columns 21 and 22, rows I – P, created by initiating the reaction with G6PD screen start solution and allowing the reaction to run till saturation, which serves as a marker for 100% G6PD activity. Drugs were dispensed into columns 5 – 20, with a final dose of 10 µM and DMSO concentration of 0.2% when the reaction mixture are added. Drugs were trialled singularly in each well and were randomly allocated by the dispensary. An illustrated summary of this plate layout is shown in Figure 2.4.2.1.

Figure 2.4.2.1 Plate layout for the G6PD HTS. Drugs were dispensed into 384- well plates, with each plate assessing 256 drugs at 10 µM concentration in a reaction volume of 40 µl. Each plate includes appropriate blanks in the first and last two columns of the plate, containing enzyme, buffer and substrate NADP+ without the substrate G6P. Reactions with enzymes incubated with 0.2% DMSO are allocated in columns 2 and 3, rows A – H, and reactions with negative control DHEA at 10 µM located in the same columns rows I – P. Reactions which receives excess NADP+ and G6P substrates are grouped in columns 21 and 22, rows A – H, and reactions which are allowed to proceed to saturation located in the same columns, rows I – P. The drugs are tested in single reactions with no technical replicates, and compounds of interest will be selected for further testing and validation.

Pretreatment of the enzyme with small molecules

An enzyme pre-treatment period, which is an incubation time whereby the enzyme interacts with a drug prior to the addition of substrates to measure enzyme activity, is often included at the start of enzyme assays. Here, we trialled two enzyme pretreatment times, 12 minutes and 15 minutes, to determine the most suitable incubation for the assay and to observe for any differences in enzyme activity with varying pretreatment periods. The reaction run time, which is the length of time the enzyme activity occurs before reaction termination, is often kept under 10 minutes (Bisswanger, 2014). Thus, reactions running for 5 and 8 minutes were trialled to determine the optimum reaction runtime to achieve distinguishable absorbance. A total of four enzyme pretreatment/reaction combinations were trialled, (i) 12 minutes enzyme-drug incubation and 8 minutes reaction; (ii) 12 minutes enzyme-drug incubation and 5 minutes reaction; (iii) 15 minutes enzyme-drug incubation and 8 minutes reaction and (iv) 15 minutes enzyme-drug incubation and 5 minutes reaction.

The assay was performed on a single 384- well plate, according to the screen plate layout (Figure 2.4.2.1) with the inclusion of appropriate controls and under standard G6PD screen protocols thus also serving as a screen validation run. The programs within the instrument required for the screen were pre-set to streamline the screen run, and all reagents were prepared as would have been required for the HTS. Furthermore, the signal to background ratio (S/B) was assessed to determine which enzyme pretreatment/reaction combination would be most suitable for enzyme activity comparison. The 0.2% DMSO control group was incubated with the parameters of the first combination, with enzyme pretreatment occurring for 15 minutes followed by 8 minutes of reaction.

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Figure 2.4.2.2 G6PD HTS optimization for enzyme pretreatment and reaction runtime. The length of time for enzyme pretreatment with small molecule was optimized in combination with the reaction runtime to determine the signal to background ratio (S/B) to achieve the optimum absorbance production for enzyme activity comparison. Enzyme was incubated for 12 or 15 minutes in the absence of treatment, and reaction allowed to run for 5 minutes or 8 minutes, resulting in the trial of four incubation time/reaction time (I/R) combination. Abbreviations: HTS, high-throughput screen; S/B, signal to background ratio; I/R, enzyme and drug incubation (minutes)/ reaction time length (minutes).

The assay blank produced a background absorbance of 0.1256 ± 0.001 AU, reaching an absorbance of 0.8614 ± 0.005 AU at saturation. The optimized G6PD activity assay produced an S/B of 6.86 when comparing the assay background to the assay saturation. In the presence of excess substrates, which was used to determine the maximum limit of the assay and to ensure the reaction saturation was not due to assay measurement limitations, an absorbance of 1.402 ± 0.019 AU was achieved, 11.16- fold over the assay background. Saturation of the reaction achieved 54.06% of this absorbance and was not limited by the mode of measurement. Addition of the G6PD enzyme inhibitor DHEA at 10 µM resulted in reduced NADPH production as measured by a decreased absorbance of 0.2315 ± 0.002 AU, which is an S/B of 1.84. This indicates a significant decrease (-53.32%, p <0.0001) in enzyme activity when compared to the 0.2% DMSO control group which has an absorbance of 0.53 ± 0.027 AU, S/B of 4.22 (Figure 2.4.2.2).

The first I/R combination of 12 minutes enzyme pretreatment and 8 minutes reaction period resulted in the production of 0.5418 ± 0.003 absorbance, S/B of 4.31, which is not significantly different to the absorbance value produced by the DMSO control group (Figure 2.4.2.2). Increasing the enzyme pre-treatment period to 15 minutes did not significantly affect the enzyme activity as well, with the absorbance measured at 0.5393 ± 0.002 AU, producing a S/B of 4.29, which was a mild decrease in activity compared to the 12 minutes enzyme pretreatment. The absorbance produced by the reactions was reduced with shortened enzyme reaction times of 5 minutes, with the 12/5 I/R combination resulting in an absorbance of 0.4305 ± 0.001 AU and S/B of 3.43, 18.77% lower than the DMSO control group absorbance. Extending the enzyme pretreatment length to 15 minutes whilst maintaining the reaction time at 5 minutes produced a mean absorbance of 0.4231 ± 0.001 AU and S/B of 3.37, which does not present any significant differences compared to the 12 minutes enzyme pretreatment group. This combination displayed a 20.17% reduction in absorbance compared to the DMSO group (Figure 2.4.2.2).

In summary, the optimized assay produced an adequate absorbance range with a S/B of 6.86, with the solvent control group having an acceptable S/B of 4.22. This control group followed the parameters of the first trial group, with 12 minutes enzyme pretreatment and 8 minutes of reaction runtime. There was no difference in absorbance produced between the two enzyme-drug incubation times and reducing the reaction runtime to 5 minutes averaged an S/B of 3.4. This is 3.36 times the assay background and is below the assay saturation by 2.03- fold. We therefore decided to proceed with a reaction combination of 15 minutes enzyme pretreatment, as this would allow for longer drug-enzyme interaction, followed by 5 minutes reaction runtime for the G6PD HTS such that the lack of difference in enzyme activity would produce an absorbance that lay at the centre of the assay absorbance range.

During the G6PD HTS screen, small molecules that increases G6PD enzyme activity will result in an elevated NADPH production rate which may be measured as increased absorbance. As the assay saturation is 2.03- fold higher than the absorbance produced by the control, this allows leeway for increased absorbance without the reaction reaching saturation. The assay will also allow for inhibitors of the enzyme to be identified, with inhibitors reducing the absorbance produced with time as seen with the DHEA negative control. The absorbance limitation of the assay is represented by the enzyme group with the inclusion of excess substrates.

G6PD HTS validation run

After determining the G6PD HTS parameters for the batch of enzyme to be used for this study, we performed a validation run. Small molecules were dispensed into allocated wells as described in the well plan (Figure 2.4.2.1), prior to the start of the assay, and the assay begins with the addition of G6PD screen substrate-enzyme mix and allowing the enzyme to interact with the small molecules for 15 minutes. The reaction was initiated with the addition of G6PD screen start solution and reaction proceeds for 5 minutes before terminating the reaction via the addition of G6PD screen stop solution. The plates were then read spectrophotometrically at 340 nm and results analysed.

A single 384- well plate was trialled for assay validation prior to the actual run with screening plates. In this screen, we pre-determined parameters that would be needed to identify hits. These were determined based on the endpoint absorbance measurement, with maximum activity determined by the relative maximum saturation, and the lack of enzyme activity from the G6P blank group. A control group where enzymes are incubated in the presence of solvent DMSO at 0.2% concentration was used to set the absorbance for normal enzyme activity, and 10 µM DHEA was used as a negative control, reducing the enzyme activity to obtain reduced absorbance.

Figure 2.4.2.3 High-throughput G6PD activator screen parameters. (A) A single 384- well plate of the optimized G6PD HTS was trialled as a screen validation prior to the screen run, with the absence of small molecules or DMSO in the treatment group. The assay had an enzyme pretreatment period of 15 minutes, with reactions proceeding for 5 minutes before terminating enzyme activity termination. DMSO control had an absorbance of 0.474 ± 0.008 AU, with the treatment group having an absorbance of 0.496 ± 0.001 AU and the presence of DHEA reduced the absorbance to 0.212 ± 0.002 AU. (B) The parameters of the G6PD HTS determined by the validation run. Inhibitors are classified as enzyme groups that have a resulting activity of 15% less than the DMSO control which had normal enzyme activity. Activators are identified as small molecules that increases enzyme activity by 15% above the DMSO control.

The assay blank, where the reaction was initiated with buffer in the absence of NADP+ produced an absorbance of 0.124 ± 0.001 AU at saturation. When the enzyme reaction containing all ingredients proceeded until all substrates were assumed to be converted to the substrate NADPH had an absorbance of 0.832 ± 0.008 AU. Using the absorbance produced by these two groups, we were able to achieve a signal to background ratio (S/B) of 6.710. The DMSO control, whereby enzyme activity is at 100% produced an absorbance of 0.474 ± 0.008 AU which is an S/B of 3.82, positioned within the absorbance range. The treatment reactions, lacking the small molecules or DMSO, produced an absorbance of 0.496 ± 0.001 AU with an S/B of 4.00. The negative control, where enzymes were pre-treated with 10 µM DHEA, produced an absorbance of 0.212 ± 0.002 AU which gives a S/B of 1.71 (Figure 2.4.2.3.A), significantly lower than the DMSO control. This is validation of the assay whereby inhibitors of the G6PD enzyme can be identified, with inhibitors reducing the absorbance produced or could be identified by comparison of the S/B.

From these results, the parameters of the G6PD HTS were identified and assigned. The assay blank was used to represent the absence of enzyme activity, where enzyme activity is at 0% and no additional absorbance is produced. The assay saturation is the absorbance limit of the reaction, and allows the maximum absorbance produced by the reaction to be known. This group is used to ensure the absorbance limit of the assay is not met following the reaction with each plate and serves as the reaction cap. The control group, where enzymes were pre-treated with 0.2% DMSO solvent to match that would be present with the small molecule treatment reactions, serves as the enzyme activity control, with enzyme activity represented at 100%. Deviations from the absorbance produced by the control group will be used to sort small molecules as drug candidates for G6PD activators, which will increase enzyme activity thus raising absorbance produced, and inhibitors which will decrease enzyme activity which reduces the absorbance produced by assay endpoint. An increase or decrease in absorbance of ± 15% from the absorbance produced by the control group was decided as the separating line for the activators or inhibitors, with reactions treated with inhibitors having <0.351 AU and activators resulting in absorbances >0.639 (Figure 2.4.2.3.B). An example of a G6PD inhibitor is as shown by the DHEA negative control group, with the absorbance produced by the inhibitor being 0.212, below the cut-off for the inhibitor classification (Figure 2.4.2.3.B).

2.4.3 G6PD HTS for enzyme activators

Following the parameters established in Results section 2.4.2 (Figure 2.4.2.3.B), small molecules that alter G6PD enzyme activity may be identified from the screen based on the deviation of endpoint absorbance from the reaction compared to the DMSO control. Each small molecule was trialled individually with no replicates, with the small molecules having been pre-administered into the allocated wells on 384- well plates as described (Figure 2.4.2.1). Assays were executed with the addition of enzyme-substrate mix into wells containing controls or drugs and allowed to incubate for 15 minutes to allow the enzyme to equilibrate and interact with the small molecules. After enzyme pretreatment, enzyme reaction was initiated with the addition of G6PD screen start solution, with reactions proceeding for 5 minutes before termination. Plates were then spun down using a centrifuge to ensure all solution were within the wells and to remove bubbles, before the reactions were read spectrophotometrically at 340 nm. The plate measurements were analysed using the Enspire in-built screening software, and the reactions of small molecules treatments sorted by degree of enzyme activation/inhibition based on endpoint absorbance. Three hundred and fifty-two small molecules were cherry- picked from the original 10,240 small molecules in the screen for further validation in triplicate, consisting of the top 298 compounds which increased the endpoint absorbance of the reactions indicating an increase in NADPH production rate, and the top 54 inhibitors of the enzyme, resulting in reduced endpoint absorbance compared to the control within the assay. The validation screen was performed under the same conditions as the G6PD HTS screen, with all compounds trialled in triplicate to isolate hit compounds which are the lead G6PD activators or inhibitors and to eliminate false positives.

Following analyses of the validation screen, small molecules were sorted based on enzyme activity determined by absorbance and 28 small molecules were identified as leads consisting of 22 activators and 6 enzyme inhibitors. These molecules were then subjected to a log dose screen, with each molecule tested for its effects on enzyme activity at 10 concentrations ranging from 39.06 nM to 20 µM. By measuring the changes to enzyme activity following treatment with small molecules at a logarithmic concentration curve, the concentration of the drug at which half-maximal response is achieved

(EC50/IC50) were determined. The results of this G6PD screen, followed by drug

88 validation and EC50 determination are tabulated below in Table 2.4.3.1 (Top enzyme activators 1 to 14), Table 2.4.3.2 (Top enzyme activators 15 to 22), and Table 2.4.3.3 (Top enzyme inhibitors).

Table 2.4.3.1 Activators of G6PD identified from the HTS G6PD screen (Compounds 1-14).

Compounds derived from the HTS screen termed ASW(1-28). EC50 values were determined using the same screening conditions as the G6PD HTS screen and are given as a mean average with standard deviation. Activities were determined by comparing mean NADPH production by G6PD enzyme in the presence of 10µM compound compared to vehicle-control with three replicates per compound tested. 89

Table 2.4.3.2 Activators of G6PD identified from the HTS G6PD screen [continued] (Compounds 15-22).

Table 2.4.3.3 Inhibitors of G6PD identified from the HTS G6PD screen (Compounds 23-28).

Compounds derived from the HTS screen termed ASW(1-28). IC50 values were determined using the same screening conditions as the G6PD HTS screen and are given as a mean average with standard deviation. Activities were determined by comparing mean NADPH production by G6PD enzyme in the presence of 10µM compound compared to vehicle-control with three replicates per compound tested.

2.5 Discussion

Activity of the enzyme glucose-6-phosphate dehydrogenase (G6PD) dictates the activity of the pentose phosphate pathway (PPP), which plays a crucial role in all aerobic organisms for processes such as the maintenance of redox homeostasis and nucleotide synthesis, as described in Chapter 1. Increasing G6PD activity and subsequently the flux of PPP could address some of the biochemical challenges faced by ageing cells, as observed in transgenic animals which over-express G6PD (Legan et al., 2008; Nobrega- Pereira et al., 2016). From these examples, we hypothesised that increasing G6PD activity via pharmacological interventions will result in improvements of lifespan and healthspan, by means of elevation of the direct and indirect antioxidant NADPH, better DNA defence and repair mechanisms and increased NAD+ availability. Therefore, the first aim of the project was to identify drugs that activates the G6PD enzyme, resulting in increased enzyme activity. A direct assay for quantitative measurement of gross G6PD activity within a sample was modified be compatible with a high-throughput assay for G6PD activity. This assay was chosen over other protocols such as the measurements of G6PD activity using indirect assays, due to the requirement of long incubation times associated with indirect assays, which may not be feasible for a high-throughput screening method as the long incubation time may affect the enzyme activity. Other assays such as the fluorescent spot test (Beutler & Halasz, 1966) and brilliant cresyl blue (Glock & McLean, 1952) quantitatively measures the G6PD enzyme activity, but are not sensitive enough for the comparison of activity between samples.

Developing the G6PD enzyme activity assay for high-throughput identification of G6PD activators from a library of small molecules required the optimization of several parameters, including optimum substrate concentrations, identifying appropriate methods for terminating enzyme activity, setting appropriate controls, assay parameters and planning the assay readout (Acker & Auld, 2014). The decision was made to have the assay optimized and conducted at room temperature, as having the reactions running at room temperature eliminates the need for thermostatting, which would shorten the experimental runtime, causing difficulties in timing, and prevent the need for thermo- controlled instrumentation. Furthermore, a considerable amount of enzyme information and experiments are conducted at this specified temperature and are deemed appropriate by The Commission on Enzymes of International Union of Biochemistry, and the 92 conditions specified for a standard assay is as described in Annex 2 of the WHO document (WHO, 1967). The target enzyme used for the purpose of this screen were also of human recombinant origin, of insectile source. Thus, we also decided to maintain the reactions at the optimum physiological pH 7.4.

As there were no known activators of the G6PD enzyme at the time of commencement of this project, 10,240 novel small molecules from the WECC-CCIA diversity library were randomly selected and tested for their ability to increase G6PD enzyme activity. These compounds were adapted from four independent vendors and have been filtered for reactive functional groups and pan-assay interfering compounds (PAINS), which often result in false positives when trialled within high-throughput screens (Dahlin et al., 2015). The drugs were also filtered for drug similarity, with compounds of 85% similarity to any other compound within the library as determined by the Tanimoto coefficient considered while building the library. Therefore, the small molecules used for the screen are not only novel, but also adhere to the Lipinski’s rule of five which favours small molecules with drug-like properties.

G6PD HTS Assay development

Each assay reaction contained G6PD enzyme, the substrates G6P and NADP+, a small molecule test compound from the drug library, or alternatively a solvent control in 50 mM Tris-HCl buffer at pH 7.4. Unlike the substrates and components of the reaction, the amount of the enzyme present within each assay reaction had to be kept as low as possible, with catalytic amounts only necessary as the enzyme was limiting and thus costly. The amount of enzyme per reaction that was suitable for this screen was determined to be 0.05 µg G6PD with 0.3 mM NADP+ trialled in the pilot experiment, and 12 nmol of NADP+ in a reaction volume of 40 µl. Assuming complete conversion to NADPH by G6PD enzyme, the total absorbance produced by the reaction was 0.663 AU (Figure 2.4.1.1) which did not reach assay saturation and produced a linear reaction rate.

Enzyme kinetics were determined for the batch of recombinant G6PD enzyme used in this screen, to identify the appropriate substrate concentration for the enzyme reaction. Km is defined as the concentration of the substrate which allows the enzyme to work at half the enzyme’s maximal velocity, Vmax. Differing variants of the G6PD enzyme, along with other variables such as enzyme storage conditions, pH, freeze-thaw cycles, impurities and contaminants could alter enzyme kinetics between protein batches. It was therefore important that the protein batch to be used in the screen was assessed and enzyme kinetics determined. To obtain the Km for G6P, measurements were taken at a saturating concentration of NADP+ whilst under varying concentrations of G6P. + Conversely, the Km of NADP was obtained under a high concentration of G6P whilst varying the concentration of NADP+.

The Km and Vmax values for the G6PD enzyme substrate G6P were determined to be 0.196 ± 0.0215 mM and 5.712 ± 0.1262 AU/hour respectively (Figure 2.4.1.2.A), producing approximately 2.856 AU/hour with NADPH being measured via absorbance + at 340 nm. The NADP substrate exhibited a lower substrate Km and mildly increased

Vmax, with Km and Vmax determined to be at 0.021 ± 0.0033 mM and 6.042 ± 0.1058

AU/hour (Figure 2.4.1.2.B), which produces an absorbance of 3.021 AU/hour. These Km + values calculated for both substrates G6P and NADP were slightly higher compared to other purified recombinant G6PD enzymes reported (Gomez-Manzo et al., 2013; Grabowska et al., 2004; Huang, Choi, Au, Au, Lam & Engel, 2008; Wang, Lam & Engel, 2005; Wang, Lam & Engel, 2006), which may be attributed to differences in the purification method, enzyme purity or glycerol content of the enzyme stock. Assuming a 1:1 complete conversion of NADP+ to NADPH within a reaction volume of 40 µl, there would be at most 0.8 nmol of NADPH in the final reaction, which will not be detectable spectrophotometrically at 340 nm (Supplementary 1). Therefore, the substrate concentrations of the G6PD enzyme assay had to be raised above the determined Km values to measurable amounts.

The equations obtained from the Lineweaver-Burk plots (Figure 2.4.1.2.C, D) enabled substrate concentrations to be substituted into the equations and the NADPH production velocity to be determined. At a G6P concentration of 0.5 mM there was a calculated velocity of 4.175 AU/hour, which is 73.09% of the maximum velocity of the enzyme. Similarly, NADP+ at 0.8 mM produces the calculated velocity of 5.905 AU/hour, reaching 97.94% the maximum velocity. Assuming all NADP+ is converted to NADPH with 0.8 mM NADP+ in the reaction, which is 32 nmol in 40 µl, this will result in an absorbance of 1.421 AU, giving a 12.18- fold signal to background ratio (S/B) (Supplementary 1), producing an adequate range for which variations in G6PD activity may be measured and compared. NADP+ is ideally kept at a lower concentration closer to Km to avoid saturation of the enzyme, as high substrate concentrations may create competition with small molecules which may bind orthosterically to the substrate active site of which most competitive inhibitors are known to interact with (Acker & Auld, 2014). However, NADP+ at 0.3 mM, which was another substrate concentration considered for the assay, produced a velocity of 5.63 AU/hour, a slight decrease of 93.38% maximum velocity whilst reducing the total assay absorbance at saturation of 0.660 AU which is a S/B of 5.66. We therefore decided to proceed with concentrations of 0.5 mM for the substrate G6PD, and 0.8 mM for the substrate NADP+.

NADPH production was recorded after a defined reaction time and following the termination of enzyme activity, ceasing further NADPH production without altering existing absorbance within the sample. Denaturing compounds such as guanidine hydrochloride (GdnHCl) and urea are often used for protein unfolding (Wingfield, 2001), and were considered as a mean of terminating enzyme activity by unfolding the protein. We prioritized our focus on GdnHCl as a potential assay stop solution for our G6PD HTS screen, as GdnHCl is generally more effective per mole as a protein denaturant than urea (Pace, 1986) and thus required a lower concentration to terminate enzyme activity. When interacting with protein, guanidium ions transiently stack and coat the hydrophobic surfaces of the protein, creating a hydrophobic effect which destabilises the protein. This coating effect and covering of the hydrophobic surfaces reduces the unfavourable exposure to water, mimicking the interactions seen in denatured or unfolded proteins (England & Haran, 2011). Thus, the direct interaction of GdnHCl with the enzyme should immediately terminate enzyme activity through unfolding of the protein, whilst not affecting NADPH which is the primary readout of the assay. 95

GdnHCl at a concentration of 2.0 M was determined to be an appropriate assay stop solution for the G6PD enzyme activity assay, with enzyme activity ceasing from when the solution was introduced into the reaction and the absorbance of existing NADPH within the solution remaining unchanged for up to 27.5 minutes after the addition of the solution (Figure 2.4.1.3). The stable absorbance post-stop solution addition is crucial for the G6PD HTS screen, as many plates will be handled in a single experiment and thus will result in a lag time between when the reaction is halted, and when the absorbance of the plate can be read spectrophotometrically. A solution of 6.0 M GdnHCl can be prepared with the solution being clear and colourless at this high concentration, with no absorbance produced at 340 nm and thus not contributing to the absorbance of the final assay reaction. Twenty microlitres of the 6.0 M GdnHCl solution were added to the reaction volume of 40 µl after the allocated reaction runtime, resulting in a working stop solution concentration of 2.0 M.

The G6PD enzyme was tested for DMSO tolerance and found to be stable, and not significantly affected by DMSO concentrations of up to 1.0 % under the standard enzyme assay conditions and at room temperature. The small molecule library was to be trialled in the G6PD HTS at a final concentration of 10 µM, for a final concentration of 0.2% DMSO solvent present in the reaction, as drug stocks are dissolved in DMSO. Therefore, no other components were required to be added to the reaction to facilitate the tolerance of the enzyme towards DMSO.

Assay parameter determination

The screening of the 10,240 small molecules was performed in 384 well plates, with a final reaction volume fixed at 40 µl per well followed by the addition of 20 µl assay stop solution to terminate the enzyme activity prior to absorbance measurement. This resulted in a total of forty 384- well plates with the drugs dispensed into allocated wells according to the well plan (Figure 2.4.2.1), and appropriate controls in place. All plates were assayed over two days, with 20 plates run per day, 10 plates each session. The conditions on the day were noted, with no difference in room temperature observed. The same protein batch were used for all assays with samples kept stored in – 30 ºC prior to use, and all solutions pre-prepared in batches filtered through a 0.45 µm filter and stored prior to use.

Every assay plate within the screen included standardized conditions and controls within the layout. Recombinant G6PD enzyme was mixed into the enzyme substrate mix in the presence of NADP+, prior to being dispensed into the allocated reaction wells as the presence of NADP+ had been found to increase the stability of the enzyme and reduces enzyme activity loss with time (Supplementary 2). Reactions were initiated with the addition of 20 µl assay mixture containing 1 mM G6P resulting in a final concentration of 0.5 mM in the reaction. An automated multidrop dispenser was used for the addition of the mixture, with manual pipetting using a multichannel pipette occurring with the addition of the initiation mixture of the excess substrate control and the saturation groups. The use of manual pipetting is usually not ideal due to errors in pipetting and the handling of small volumes. However, time constraints demanded a compromise which required the use of both manual and automated initiation of the reaction.

Every assay plate consisted of reaction blanks on either side of the plate to ensure there were no variations in absorbance due to the orientation of the plate and to also serve as the reaction baseline. The positive control, whereby the enzyme reaction was allowed to proceed with increased length of incubation time, was used to determine the relative degree of saturation, where absorbance is at maximum for the substrate concentrations provided within the reaction. This is also to ensure that the saturation limit of the reaction is not reached by any of the treatments tested and the controls. Together, these controls allowed the absorbance range of the assay to be determined. Reactions with DMSO of concentration 0.2% were used to represent normal enzyme activity, where activity are represented as 100% for comparison of deviations of activity in the presence of small molecules. A fixed enzyme-drug incubation time, known as the enzyme pretreatment, and reaction runtime was determined such that the absorbance produced by the DMSO control group was within the middle of the baseline and saturation absorbance range.

The production of NADPH by recombinant G6PD enzyme in vitro was noted to reduce and halt after a reaction period, with the NADPH levels, measured by the absorbance at 340nm, plateauing and reaching a saturation limit with given time as seen in Figure 2.4.1.1. This is possibly due to exhaustion of the enzyme substrates G6P or NADP+ in the reaction mixture. Another possible explanation for the observation is that the enzymatic product, NADPH, is also known to inhibit the activity of the G6PD enzyme (Glaser & Brown, 1955). An 8 minute reaction time was considered for the enzyme reaction, which would provide a good absorbance range for the enzyme assay but would increase the overall assay runtime which is ideally kept at a minimum due to time constraints. Therefore, the reaction time set for the enzyme activity assay was allocated 5 minutes, whereby the NADPH production rate is linear which occurs typically within the first 10 minutes of the enzyme reaction.

Prior to reaction initiation, the enzyme were allowed to incubate in the presence of the small molecule treatment for 15 minutes to equilibrate the enzyme to the drugs. Two enzyme pretreatment times were trialled, with little to no difference observed between the incubation times for 12 minutes and 15 minutes (Figure 2.4.2.2). We decided to proceed with the 15 minutes pretreatment time as this would enable more time for the drugs to interact with the enzyme, without the risk of losing much enzyme activity with the incubation time. Under the G6PD screen validation run, the combination of 15 minutes enzyme pretreatment and 5 minutes reaction runtime produced an adequate absorbance of 0.474 AU, lying 3.82- fold above the baseline and 1.76- fold below the assay saturation (Figure 2.4.2.3.A). Small molecules which increased G6PD enzyme activity would elevate endpoint absorbance towards the assay saturation, whilst inhibitors will reduce the endpoint absorbance, deviating towards the baseline. Therefore, the combination of enzyme pretreatment and reaction runtime in the optimized assay are able to accommodate for these changes in absorbance, allowing enzyme activators and inhibitors to be determined.

The small molecules were sorted as enzyme activators or inhibitors based on the absorbance produced in comparison to the DMSO control group, with deviations of 15% absorbance from the endpoint absorbance of the control group being appointed as the cut- off for the classification of the drugs (Figure 2.4.2.3.B). A previously described inhibitor of G6PD, DHEA (Gordon, Mackow & Levy, 1995) was used as a negative control in every assay plate in the screen. Incubation of the enzyme in the presence of DHEA at 10 µM reduced the endpoint absorbance of the assay to 0.212 AU, which is below the pre- determined cut-off for inhibitors in the assay (Figure 2.4.2.3.B). With all these optimizations and parameters in place, the screen was ready for commencement.

G6PD enzyme small molecule HITS

The G6PD screen for the trial for small molecules which increase or decrease G6PD enzyme activity was run according to parameters and conditions which were established in Results section 2.4.2 G6PD HTS validation. The screen was performed at room temperature, which was 21ºC at the time of the experiment within the room where the screen was performed. Plate measurements were analysed in-house using the Children’s Cancer Institute Australia (CCIA) Drug Discovery Centre screening software, resulting in 352 small molecules with the largest deviations from the control endpoint absorbance cherry-picked and subjected to validation in triplicate to process out false positives. These small molecules consisted of 298 potential G6PD activating compounds and 54 G6PD inhibiting candidates, as the priority of the screen was for the identification of G6PD activators.

G6PD inhibitors were included within the validation screen to be used as a negative control for future experimentation. Furthermore, G6PD inhibitors may prove useful for further exploration, as inhibitors of G6PD are commonly used as therapeutics for specific diseases and pathologies as elaborated in Chapter 1. Commonly used G6PD inhibitors such as 6-aminonicotinamide and DHEA lacks target enzyme specificity, with 6-aminonicotinamide having toxic side effects, whilst DHEA causes pleiotropic effects such as growth inhibition that is not related to the inhibition of G6PD (Biaglow, Ayene, Koch, Donahue, Stamato & Tuttle, 2000; Gupte, 2008; Ng, Wang, Lee & Hu, 1999; Paulin, Meloche, Jacob, Bisserier, Courboulin & Bonnet, 2011). Due to the lack of specificity of these inhibitors, identification of G6PD inhibitors may be useful as potential pharmacological treatments for G6PD-related diseases. G6PD inhibition may prove useful for synergistic effects when used in combination with chemotherapy and in cancer treatment (Catanzaro et al., 2015; Jones & Schulze, 2012; Ju et al., 2017; Li, Fath, Scarbrough, Watson & Spitz, 2015; Mele et al., 2018). Furthermore, there are no specific G6PD inhibitors in clinical settings to date (Mele et al., 2018), despite the PPP having been identified as a target for cancer therapy (Cho, Cha, Kim, Kim & Yook, 2018; Jones & Schulze, 2012; Pavlova & Thompson, 2016).

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Twenty-eight small molecules were isolated from the validation screen, with 22 of the compounds being activators of the G6PD enzyme and 6 being G6PD enzyme inhibitors. These are novel small molecules and were given the terms ASW01 – ASW28 for ease of naming and categorization, with the small molecules sorted numerically via the magnitude of change to enzyme activity. The EC50/IC50 of these 28 compounds were also determined via subjecting the compounds to a 10- dose log screen, with concentrations varying from 39.06 nM to 20 µM being tested. All small molecule details and changes to enzyme activity are summarized in Table 2.4.3.1 and Table 2.4.3.2, with with the log dose curves for EC50/IC50 determination available in Supplementary figures

4 – 8. The EC50 for the compound ASW10 could not be determined due to the lack of absorbance produced by the group. This could have been due to a pipetting error, with the enzyme or substrates not having been included within the reaction or may be due to insufficient enzyme activity detected in the presence of the drug. However, in the G6PD HTS and validation screen, the compound ASW10 exhibited qualities of being an activator of the G6PD enzyme.

Compound ASW01 exhibited the highest increase in G6PD enzyme activity when present at 10 µM concentration within the enzyme assay reaction, with an average increase in enzyme activity of 167.88% when trialled in triplicate with an EC50 of 6.43 ± 0.09 µM. Compound ASW22 exhibited the least increase in enzyme activity out of the 22 activators isolated from the validation screen, with an increase of activity to 133.68% compared to the control with an EC50 of 7.25 ± 1.26 µM observed in vitro. There were no known small molecule activators of the G6PD enzyme at the commencement of this project in 2015, thus the comparative efficacy of the small molecules isolated from the screen could not be assessed. Recently, a G6PD activator known as AG1 has since been discovered which increased wild-type G6PD enzyme activity, restores the activity of deficiency variants Canton, A-- and several others (Hwang et al., 2018). This compound increased enzyme activity of recombinant wild-type G6PD up to 20% over basal activity, and G6PD_Canton variant by up to 170% with drug EC50 reported to be 3 µM (Hwang et al., 2018). This is a modest increase in wild-type enzyme activity compared to the observed increase in activity by the ASW small molecules activators isolated from the G6PD HTS, with at least 133.68% increase in activity observed compared to the solvent control.

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Despite AG1 having mild efficacy in the activation of wild-type G6PD, Hwang et al. showed that the molecule also decreases oxidative stress in zebrafish, cells and stress- induced erythrocytes, now known to occur by promoting G6PD monomer oligomerization (Raub et al., 2019), with effects on animal survival and lifespan remaining to be elucidated. There are some evidence showing the elevation of G6PD activity having beneficial effects on longevity and survival in animals such as in fruit flies (Legan et al., 2008) and mice (Nobrega-Pereira et al., 2016) as further elaborated in Section 1.4.5 Ageing: healthspan and lifespan. Thus, the activators of G6PD identified from this HTS were tested for similar modes of action to better characterize these molecules (Chapter 3), with priority for changes occurring with animal lifespan being elaborated in Chapter 4.

Compounds ASW23 – ASW28 were identified from the G6PD HTS as G6PD- inhibiting small molecules, with compound ASW23 having the highest G6PD enzyme activity inhibition, reducing enzyme activity to that of 18.84% normal enzyme activity, whilst ASW28 exhibited the least enzyme inhibition of the six isolated compounds, with a reduction of activity to 35.83% of normal activity. The IC50 of the molecules ranged from 1.04 µM to 10.31 µM, with compound ASW24 exhibiting the highest drug potency of the identified G6PD inhibitors as summarized in Table 2.4.3.3 with log dose graphs for IC50 determination for the inhibitors available in Supplementary 8. With the IC50 of <10.31 µM, the inhibitors identified from this work are approximately 100 -fold more potent compared to the known G6PD inhibitor DHEA which exhibits an IC50 of approximately 300 µM and 6-aminonicotinamide which has an IC50 of >3 mM (Preuss et al., 2013). The small molecule inhibitors are however similar in potency and efficacy to those identified recently (Mele et al., 2018; Preuss et al., 2013). The IC50 of polydatin, a glucoside of resveratrol found to inhibit the G6PD enzyme in a dose-dependent manner was determined to be 22 µM (Mele et al., 2018), whilst a similar HTS performed for the identification of novel inhibitors of G6PD yielded compounds with IC50 values ranging from 0.4 – 2.7 µM (Preuss et al., 2013). Differences in experimental methods such as drug-enzyme preincubation times and substrate concentrations may create variability in

IC50 determination. Lead compounds identified from the HTS were subjected to further experimentation to determine the mode of action on the G6PD enzyme, with focus on the changes to enzyme stability. These findings are elaborated on in Chapter 3.

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Chapter 3 Characterising ASW Compounds

Characterising ASW Compounds

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3.1 Introduction

The G6PD high-throughput screen (HTS) described in Chapter 2 resulted in the identification of a series of small molecules which increased G6PD enzyme activity. In this chapter, these small molecules were further explored to determine the mode of action by which these compounds alter enzyme activity with a focus on changes to enzyme stability. Most ASW compounds were found to increase G6PD enzyme activity by preventing enzyme instability, resulting in a reduction in the loss of enzyme activity that occurs in wild type G6PD enzyme at room temperature. Following this observation, we hypothesised that the ability of these compounds to stabilise enzyme activity could be useful in the treatment of G6PD deficiency, whereby the mutations within the G6PD gene result in increased enzyme instability, reduced specific activity or both (Beutler & Vulliamy, 2002; Luzzatto, 2006; Naylor et al., 1996; Vulliamy, Mason & Luzzatto, 1992).

The effects of these ASW compounds on enzyme kinetics of recombinant G6PD enzyme treated were also determined by varying enzyme substrate concentrations and the lead molecules were subjected to assessment to determine antioxidant capacity of the drugs. Compounds which stabilised G6PD enzyme activity could prevent loss of activity with time, due to inherent enzyme instability, potentially being effective in the therapeutic treatment of patients with pathological variants of G6PD. To test this, recombinant protein of several common G6PD deficiency variants were produced to test whether these compounds could increase enzyme activity. Receptor-ligand docking simulations were also used to elucidate the interactions of the drugs with the G6PD enzyme to better understand the mechanism of action of the small molecules, showing favourable binding to the NADP+ substrate binding region of the enzyme to improve the stability of the enzyme.

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3.2 Materials

3.2.1 Materials and reagents

Materials

β-Nicotinamide adenine dinucleotide phosphate hydrate [Sigma: N5755], D-Glucose 6- phosphate disodium salt hydrate [Sigma: G7250], β-Nicotinamide adenine dinucleotide 2′-phosphate reduced tetrasodium salt hydrate [Sigma: N7505], Tris (Hydromethyl Methylamine) [AJAX: 2311], Trizma® hydrochloride [Sigma: T5941], 6- Phosphogluconic acid trisodium salt [Sigma: P7877], trans-dehydroandrosterone [Sigma: D4000], Dimethyl sulfoxide [Sigma: D5879], Copper (II) chloride LR dihydrate (Chem- supply: CL004), Acetic acid (AJAX: AJA1 PL), Ammonium hydroxide (AJAX: A43 GL), Neocuproine (Sigma: N1501), Dimethyl sulfoxide (Sigma: D8418), Trolox ((±)-6- Hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid) (Sigma: 238813), N-Acetyl-L- Cysteine (LKT: A0918), (+)-Sodium L-ascorbate (Sigma: A4034), Recombinant human Glucose-6-Phosphate Dehydrogenase enzyme [Abcam: ab126671], Sodium chloride [AJAX: 465], Imidazole [Sigma: 56750], 2-Mercaptoethanol [Sigma: M3701], cOmplete EDTA-free Protease inhibitor cocktail tablets [Roche: 05056489001], Peptone [Sigma: 70178], Potassium chloride [AJAX: 383], Magnesium chloride [Sigma: M8266], D-(+)- Glucose [Sigma: G8270], Yeast extract powder [Sigma: Y1625], TALON Superflow cobalt resin [Clontech Laboratories, Inc.], Pierce™ ECL Western Blotting Substrate [Thermo Scientific™: 32106].

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Reagents

SOC media [2% Peptone, 0.5% yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 mM

MgCl2, 20mM Glucose] (adapted from Cold Spring Harbour protocol (2012))

Bacterial cell lysis buffer [50 mM Tris-HCl, 200 mM NaCl, 5 mM Imidazole, 5 mM 2-Mercaptoethanol, protease inhibitor, pH 8.0]

Protein purification equilibration/wash buffer [50 mM Tris-HCl, 200 mM NaCl, 10 mM Imidazole, pH 8.0]

Protein purification elution buffer [50 mM Tris-HCl, 200 mM NaCl, 200 mM Imidazole, pH 8.0]

3.2.2 Instruments

SpectraMax M Multi-mode microplate reader (Molecular Devices), EnSpire® EnVision Multimode Plate Reader (PerkinElmer), Bacterial incubator (Thermoline, NSW, Australia), Shaking incubator (Ratek, Vic, Australia), Branson sonifier 250 (Model 102C, Fisher Scientific), BIOVIA Discovery Studio 4.1 Software.

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3.3 Experimental Methods

3.3.1 G6PD enzyme activity assay

G6PD activity was determined as described previously (Tian et al., 1998), by monitoring the reduction of NADP+ at 340 nm. Each reaction mixture consisted of 50 mM Tris-HCl (pH 7.4), 0.3 mM NADP+, and 0.05 µg G6PD enzyme. The enzymes were incubated in the presence of compounds, with reactions being initiated with the addition of G6P to a final reaction concentration of 0.5 mM and activity measured using a spectrophotometer at room temperature (24 °C). NADPH production was measured at 340 nm every 20 seconds for at least 20 minutes, and the rate of NADPH production determined as a unit of AU/hour from the initial 2 – 5 minutes of the initial reaction slope.

3.3.2 G6PD kinetic studies

Changes in Km and Vmax of drug-treated enzymes were determined using Lineweaver- Burk curves (Lineweaver & Burk, 1934). These were obtained by titrating NADP+ (0 – 800 µM) while keeping constant saturating concentration of G6P. This was performed similarly for G6P with a fixed saturating concentration of NADP+ and varying G6P concentrations (0 – 500 µM). All kinetic studies were performed at 24 ºC and at optimal pH (50 mM Tris-HCl, pH 7.4), with absorbance of the reaction measured at 340 nm every

20 seconds, and the initial slope, Km and Vmax values were calculated by nonlinear regression using Graphpad Prism 7 software (Graphpad, San Diego, CA).

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3.3.3 Construction, design and site-directed mutagenesis of recombinant G6PD

The plasmid encoding wild-type G6PD protein within the pSJ3 vector was received as a gift from Dan Ye (Key Laboratory of Molecular Medicine of Ministry of Education and Institutes of Biomedical Sciences, Shanghai Medical College, College of Life Science, Fudan University, Shanghai, China). This was amplified using Q5® high-fidelity DNA polymerase (New England Biolabs) with G6PD forward and reverse primers containing single base changes corresponding to the target G6PD mutation variant (Table 3.3.1), and according to the QuikChange® Site-Directed Mutagenesis Kit instructions (Stratagene). PCR products were digested with Dpn I restriction enzyme at 37 ºC for 1 hour to digest the parental DNA, and the reaction mixture were transformed into competent NEB® 5- alpha E. coli cells (New England Biolabs). Plasmids were isolated from the cells using GenEluteTM Plasmid Miniprep Kit (Sigma-Aldrich), and all constructs were verified by DNA sequencing.

Table 3.3.3.1 List of primers used for sequence confirmation and site-directed mutagenesis

Primers were designed according to the mutagenic primer design guidelines of QuikChange® Site-Directed Mutagenesis Kit (Stratagene).

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3.3.4 Bacterial transformation procedure

Competent BL21 (DE3) cells (Novagen) were transformed with plasmid pRIL (Stratagene) and subsequently plasmid pBB550 (RRID: Addgene_27396) encoding genes for chaperone protein expression. Cells were then transformed with plasmid carrying the constructed gene for target G6PD protein as described in Section 3.3.3. Briefly, 2 µl plasmid mixture were introduced to competent cells, and transformed via heat-shock at 42 ºC for 40 seconds. 100 µl SOC media were then added to the sample and the mixture cultured for 30 minutes at 37 ºC in a shaking incubator before being plated on LB agar containing antibiotics. The plates were incubated overnight at 37 ºC, and single colonies were isolated, cultured and stored at -80 ºC in 20% glycerol. Cells were cultured in the presence of appropriate antibiotics for the maintenance of all plasmids, as detailed in Table 3.3.2.

Table 3.3.4.1 Plasmids and their corresponding antibiotics for maintenance.

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3.3.5 Computational molecular docking

The binding affinity of small molecules to the G6PD enzyme were conducted using Receptor-ligand docking simulations on Discovery studio 4.1 (DS4.1, BIOVIA, formerly Accelrys) running on a Windows 7 operating system. Simulations were performed on the three-dimensional (3D) crystal structure of G6PD monomer for NADP+ structural (PDB: 1QKI) (Au, Gover, Lam & Adams, 2000) and catalytic site (PDB: 2BH9) retrieved from the Protein Data Bank (www.rcsb.org). The structures were prepared by optimization using the Minimization protocols of DS4.1. A binding-sphere was subsequently defined at substrate binding regions of the enzyme prior to docking the compounds. By using the Prepare Ligands protocol, the compounds were generated and were subjected to molecular docking studies. The binding affinity of the small molecules were compared to that of the optimized substrate of NADP+ and represented as the Goldscore binding affinity, with the amino acid interactions identified using the best scoring ligand docking pose.

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3.3.6 Expression and purification of G6PD enzymes

Overnight culture of BL21 (DE3) cells expressing chaperones, G6PD and its variants (as described in methods Section 3.3.3) were used to inoculate a larger culture of Luria

Bertani (LB) medium at a starting cell density of 0.05 OD600. Bacteria were cultured at 37 ºC until a cell optical density of approximately 0.4 was reached before the cultures were cooled to 16 ºC. Gene expression was induced with 200 µM IPTG addition and grown for 16 - 20 hours before harvesting the bacteria by centrifugation at 2500 g for 20 minutes, washing the pellet once with H2O and storing in -30 ºC. Soluble and insoluble fractions were analysed by SDS-PAGE and western blotting for G6PD protein.

1 g of cell pellet was resuspended in 16 ml bacterial cell lysis buffer, and lysed via sonication at 30% amplitude output, 50% duty cycle, 20 pulses on ice with sonication cycle repeated three times, before being centrifuged at 16’000 g at 4 ºC for 20 minutes. Supernatant was collected and passed through a 0.22 µM filter before being subjected to gravity-flow column protein purification using HisTALON™ resin (Clontech Laboratories, Inc.) pre-equilibrated with protein purification equilibration buffer. Unbound protein were washed off the resin with 20 column volume of wash buffer, and protein eluted with 5 column volume of elution buffer. The protein were dialysed and concentrated using Amicon centrifugal filter unit (Merck), and protein concentration were determined by Bradford assay. Protein were handled at 4 ºC or on ice throughout the purification procedure.

3.3.7 SDS-PAGE and Western blot analysis

Samples were normalised by protein concentration and protein were size-fractionated electrophoretically using commercial 4-20% gradient SDS-polyacrylamide gel electrophoresis or 8% SDS-polyacrylamide gel electrophoresis made using Bio-rad hand- cast system. The protein were then transferred to PVDF membranes blocked with 2.5% skim-milk solution and the membranes were incubated in primary antibody G6PD (1:1000, Sigma: HPA000247) followed by the appropriate secondary antibody before being visualised with an HRP-ECL detection system.

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3.4 Results

3.4.1 ASW compounds stabilise, rather than activate G6PD enzyme activity

The effects of ASW compounds on G6PD enzyme activity were studied with a reduced enzyme pretreatment timelength of 5 minutes to assess the importance of the drug-enzyme incubation and to determine if the drugs were able to increase enzyme activity without the need for pre-incubation. This was conducted alongside the G6PD enzyme activity assay under the same experimental parameters and conditions as the screen assay, with enzyme pre-treatment kept consistent with 15 minutes drug-enzyme incubation as a means of validating the compounds and the previous results obtained from the initial screen. Of the 28 lead compounds identified from the G6PD HTS, we managed to procure 20 small molecules from drug and chemical distributors with the other 8 being unavailable at the time of the experiment. All other parameters of the assay were unchanged to that used in the screen, with substrate concentrations kept at 0.8 mM NADP+, 0.5 mM G6P and the small molecules trialled on 0.05 µg recombinant G6PD enzyme. All compounds were trialled at 10 µM concentration, with the DMSO solvent kept consistent across all groups at 0.1%.

The presence of the G6PD inhibitor DHEA significantly reduced enzyme activity with both enzyme pretreatment durations, reducing the enzyme activity rate from 2.592 ± 0.017 AU/hour to 1.782 ± 0.218 AU/hour (-13.25%, p = 0.01) following 5 minutes of enzyme pretreatment and from 1.348 ± 0.017 AU/hour to 0.736 ± 0.218 AU/hour (- 45.42%, p = 0.0001) activity loss within a 15 minute duration. Only two of the twenty compound treatments altered enzyme activity following 5 minutes of pretreatment, with ASW13 significantly raising enzyme activity by 33.02% (p = 0.039) and ASW25 resulting in decreased enzyme activity by 47.84% (p = 0.0006). The other G6PD activators and inhibitors trialled in the assay did not affect G6PD enzyme activity, despite having been isolated from the screen for their capabilities of altering enzyme activity (Figure 3.4.1.1.A).

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Figure 3.4.1.1 Effect of ASW compounds on G6PD enzyme activity with varied lengths of enzyme pretreatment. (A) G6PD enzyme activity following 5 minutes incubation with ASW compounds. The G6PD inhibitor DHEA was used as a negative control. Compound ASW13 significantly increased the G6PD enzyme activity following 5 minutes of pretreatment with the enzyme, while other G6PD activators trialled in this experiment did not. From six G6PD inhibitors trialled, only ASW25 significantly reduced activity. (B) Effect of 15 minutes enzyme pretreatment with ASW compounds on G6PD enzyme activity. Enzyme pretreatment with several putative small molecule activators of G6PD increased enzyme activity, with ASW03, ASW04, ASW06, ASW07, ASW08, ASW13, ASW21 and ASW22 all significantly increasing G6PD enzyme activity. Statistical analyses were performed using One-way ANOVA, error bars are S.E.M.

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When G6PD enzyme was treated with small molecules for the increased timelength of 15 minutes, treatments with the G6PD-stabilising ASW compounds ASW03, ASW04, ASW06, ASW07, ASW08, ASW13, ASW21 and ASW22 resulted in significantly increased G6PD enzyme activity compared to the control, with more compounds responding than when incubated for 5 minutes. Compound ASW22 treatment resulted in the largest increase in enzyme activity, with an absorbance production rate of 2.778 ± 0.012 AU/hour (p = 0.0001), 106.08% above that of the control group. Compounds ASW07, ASW06 and ASW13 increased enzyme activity by 93.03% (2.602 ± 0.055 AU/hour, p = 0.0001), 86.50% (2.514 ± 0.173 AU/hour, p = 0.0001) and 84.94% (2.493 ± 0.031 AU/hour, p = 0.0001) respectively.

Compounds ASW03 and ASW04 were more modest in the increase of enzyme activity, with ASW03 increasing enzyme activity by 71.74 % (2.315 ± 0.030 AU/hour, p = 0.0001) and ASW04 raising enzyme activity by 55.56% (2.097 ± 0.198 AU/hour, p = 0.0001). ASW08 and ASW21 also increased enzyme activity by 55.42% (2.095 ± 0.030 AU/hour, p = 0.0001) and 44.88% (1.953 ± 0.053 AU/hour, p = 0.002). The inhibitors of G6PD ASW24 and ASW25 significantly decreased enzyme activity by 43.81% (0.757 ± 0.07 AU/hour, p = 0.003) and 76.45% (0.318 ± 0.032 AU/hour, p = 0.0001) respectively. Other compounds identified from the screen to alter G6PD activity including compounds ASW01, ASW02, ASW12, ASW15, ASW19, ASW20, ASW23, ASW26, ASW27 and ASW28 did not significantly alter enzyme activity (Figure 3.4.1.1.B).

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It was also noted that increasing the enzyme pretreatment period from 5 to 15 minutes resulted in a decrease in mean G6PD activity, from 2.592 ± 0.288 AU/hour to 1.348 ± 0.017 AU/hour when comparing the control groups of both experiments (Figure 3.4.1.1.A and B), suggesting a loss of enzyme activity with time when the enzyme was incubated in vitro. This suggested instability of the G6PD enzyme during the experimental procedure, with ASW compound treatments preventing the time-dependent loss of enzyme activity, instead of increasing baseline enzyme activity. Comparing between the two enzyme pretreatment timeframes, the longer drug-enzyme incubation period of 15 minutes was 10 minutes longer than the 5 minutes duration, which resulted in increased enzyme activity loss within that time. The presence of several of the ASW compounds such as ASW03, ASW04, ASW06, ASW07, ASW08, ASW13, ASW21 and ASW22 maintained the enzyme activity, exhibited through the raised G6PD activity following in the presence of the compounds.

We next performed an experiment to assess enzyme stability measured by the changes to enzyme activity with increasing incubation times in vitro, under the same assay parameters. Enzyme activity decreased with longer enzyme pretreatment times, which in this assay was represented by the incubation time, that is, the length of time the enzyme was incubated in the wells of the assay plate kept at room temperature and away from light. Following a 12 minute incubation time, G6PD enzyme activity was 20.77 ± 0.318 µmol/min/mg. This activity was inversely proportional to the enzyme incubation time, with increasing incubation resulting in decreased enzyme activity. Increasing the enzyme incubation time to 23 minutes resulted in a reduced enzyme activity of 11.00 ± 0.450 µmol/min/mg and incubation time to 35 minutes decreasing enzyme activity to 4.144 ± 0.225 µmol/min/mg (Figure 3.4.1.2).

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H u _ G 6 P D _ W T A c tiv ity w ith tim e

2 5

o i

) 2 0

/ d

n 1 5

l 1 0

µ D

( 5

A N 0 1 2 2 3 3 5

In c u b a tio n tim e (m in u te s )

Figure 3.4.1.2 Recombinant human wild-type G6PD loses activity over time in vitro. G6PD enzyme were dispensed into wells of 384- well plate and allowed to incubate for varying lengths of time to assess enzyme stability in vitro. Incubation for 12 minutes resulted in enzyme activity of 20.77 ± 0.318 µmol/min/mg with enzyme activity decreasing with increased incubation times. Raising the incubation time to 23 minutes resulted in reduced enzyme activity of 11.00 ± 0.450 µmol/min/mg and 35 minutes down to 4.144 ± 0.225 µmol/min/mg. A shorter incubation time was not available due to time required from reaction initiation to absorbance measurement.

These observations showed that recombinant human wild-type G6PD protein is unstable in vitro, with enzyme activity decreasing over time, and the ASW compounds initially predicted to be activators of the G6PD enzyme were revealed to function more as enzyme stabilisers. Subsequent G6PD enzyme activity assays for small molecule testing were therefore performed with 30 minutes enzyme pretreatment prior to initiating the reactions, allowing enzyme activity loss during the pretreatment period and measuring activity after to assess the activity preservation by the treatments.

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All twenty ASW compounds were subjected to a modified G6PD enzyme activity assay for the assessment of enzyme stabilisation capacity of the small molecules (Figure 3.4.1.3 and Supplementary 3). This was measured by the changes to enzyme activity from an initial timepoint to an incubation period of 30 minutes, whereby enzyme destabilisation occurred, and enzyme activity was reduced. The vehicle control group consisted of enzyme treated with 0.1% solvent DMSO, with the enzyme activity at time 0 representing normal enzyme activity of 100%. All results were collated from at least three independent experiments under identical conditions. There were no significant changes to enzyme activity in the absence of drug-enzyme incubation or enzyme activity loss at time 0, with the exception of compound ASW25-treated enzyme which exhibited a significant decrease in activity of 65.5% (p = 0.0002) compared to the control (Figure 3.4.1.3). There was also a slight decrease in enzyme activity with the ASW24-treated enzyme, but with no statistical significance.

Thirty minutes of incubation at room temperature decreased the enzyme activity of the control group by 63.25% compared to time zero, indicating a loss of activity by enzyme destabilisation. Several G6PD-stabilising compounds including ASW03, ASW13, ASW21 and ASW22 prevented this activity loss with the enzyme activity being maintained at 91.76% (p = 0.0009), 84.68% (p = 0.0122), 92.38% (p = 0.0023) and 85.45% (p = 0.0045) respectively, which was significantly raised compared to the DMSO control at the 30 minutes timepoint. Other G6PD stabilisers trialled for preservation of enzyme activity were compounds ASW04, ASW06, ASW07 and ASW08, which exhibited trends towards maintaining enzyme activity and preventing of loss of activity, but without statistical significance established when comparing enzyme activity at timepoint 30 minutes to the control group. Enzymes treated with the inhibitors ASW24 and ASW25 exhibited a mild decrease in activity, with no statistically significant difference compared to the control group at the 30 min timepoint (Figure 3.4.1.3). Other ASW compounds derived from the G6PD HTS were also trialled under the same experimental conditions to assess if the compounds also stabilised the enzyme as those presented in Figure 3.4.1.3, but no changes to enzyme activity were observed. These compounds were ASW01, ASW02, ASW12, ASW15, ASW19, ASW20, ASW23, ASW26, ASW27 and ASW28 (Supplementary 3).

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A S W C o m p o u n d s sta b iliz e s r e c o m b in a n t h u m a n w ild -ty p e G 6 P D e n z y m e

1 5 0

* ** D M S O C o n tr o l )

l ** A S W 0 3

r t

n A S W 0 4

o ***

c 1 0 0

A S W 0 6

o t

A S W 0 7

(

y A S W 0 8

v i

t A S W 1 3 c

A 5 0 A S W 2 1

D *** P

6 A S W 2 2 G A S W 2 4

A S W 2 5

te te u u in in m m 0 0 3

Figure 3.4.1.3 Lead G6PD activators function as enzyme stabilisers preventing loss of enzyme activity over time. Several lead ASW compounds derived from the G6PD HTS were trialled for enzyme stabilising properties by comparing enzyme activity at time 0 and 30 minutes, after which G6PD enzyme experiences a decrease in enzyme activity, with DMSO control at time 0 representing 100% enzyme activity. G6PD enzyme in the presence of ASW compounds showed no significant difference in G6PD activity compared to the control gorup at time 0, with the exception of compound ASW25-treated enzyme, which exhibited a decreased activity of 65.5% (p = 0.0002) compared to the control DMSO treatment despite the lack of enzyme pretreatment. G6PD enzyme activity decreased by 63.25% after 30 minutes of incubation within the control group, indicating enzyme destabilisation. Compounds ASW03, ASW13, ASW21 and ASW22 prevented activity loss with the enzyme activity maintained at 91.76% (p = 0.0009), 84.68% (p = 0.0122), 92.38% (p = 0.0023) and 85.45% (p = 0.0045) respectively which were significantly raised when compared to the DMSO control at the 30 minutes timepoint. Results were summarized and graphed from at least three independent experiments.

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3.4.2 Lead G6PD-stabilising ASW compounds predicted to affect enzyme dimerization resulting in altered G6PD enzyme kinetics

In silico molecular docking simulations were used to predict the interactions of enzyme-stabilising ASW compounds from the G6PD enzyme activity assays as well as the binding affinities of the drugs to specific regions on the enzyme. Protein-ligand docking simulations within the Biovia Discovery studio 4.1 software were used to score the binding affinities of the small molecules to the enzyme, with a focus on common molecular interactions areas such as the substrate binding sites. The NADP+ binding regions were of interest as possible targets of the ASW small molecules, as the G6PD enzyme consists of two NADP+ binding sites: a catalytic NADP+ site involved in catalysis, and a structural NADP+ binding site securing the long-term stability of the enzyme (Wang, Chan, Lam & Engel, 2008b). As the ASW small molecules exhibited enzyme stabilising properties when trialled in the G6PD enzyme activity assays, resulting in the maintenance and preservation of enzyme activity, there could be interactions occurring at the structural NADP+ binding region following the ASW drug treatments leading to enzyme stabilisation.

Using the Receptor-ligand docking simulation within the DS4.1 software package, we were able to predict the amino acid interactions by a specified ligand, determined via the optimum ligand pose within the assigned binding-sphere which had been set at both the catalytic and structural NADP+ binding sites located on the enzyme. The binding affinity of the small molecules to the NADP+ binding sites were determined via Goldscore analysis of the protein-ligand interaction under 100 docking simulation poses, with the best docking conformation used to analyse amino acid interactions. The binding affinities of the ASW small molecules to the NADP+ binding pockets of the enzyme were represented by Goldscore fitness, summarized in Table 3.4.2.1.

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Table 3.4.2.1 Predicted compound-enzyme binding affinity of ASW compounds to G6PD NADP+ binding sites

Drug-enzyme binding affinity are represented in Goldscore fitness, as determined by in silico docking studies. G6PD enzyme activity were obtained from the G6PD HTS analyses, with activity having been determined following 15 minutes of enzyme-drug pretreatment and normalised to the activity of the DMSO control. Small molecules were fitted against the NADP+ binding regions of the three-dimensional (3D) crystal structure of G6PD monomer for NADP+ structural (PDB: 1QKI) (Au, Gover, Lam & Adams, 2000) and catalytic site (PDB: 2BH9) retrieved from the Protein Data Bank (www.rcsb.org).

The substrate NADP+ was used as a positive control for the comparison of ligand binding affinities to the NADP+ binding sites, providing a Goldscore fitness of 82.07 for the structural binding site, and the slightly reduced score of 68.96 for the catalytic binding site indicating preference for substrate binding at the former NADP+ binding region. The binding affinities obtained for ASW small molecules ranged from 19.30 to a top of 66.40 for the structural NADP+ site and 10.60 to 63.22 for the catalytic NADP+ site (Table 3.4.2.1). Despite being of lower score than the NADP+ control, these scores are within range for small molecules which are smaller in size compared to the structure of NADP+ resulting in a lowered score due to fewer molecular interactions. There were no correlation between the changes of G6PD enzyme activity following drug treatment and the binding affinities of the drug to the enzyme, with enzyme activity being represented by the results obtained from the G6PD HTS.

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The compound ASW18 exhibited the highest binding affinity of the trialled small molecules for the structural NADP+ binding region of the enzyme, with a score of 48.81 for the catalytic binding site. Results from the G6PD high-throughput screen indicated ASW18 to have a modest improvement in enzyme activity, although this drug had not been further investigated due to limitations of drug availability. Compound ASW12 did not aid in the stabilisation of G6PD enzyme when trialled in vitro on recombinant G6PD, but scored highest in binding affinity for the catalytic region with good predicted binding affinity for the structural NADP+ binding region. All trialled ASW small molecules had a binding affinity of Goldscore 45.0 and higher, which is positive for an interaction to the structural site, with the exception of the two G6PD stabilising small molecules ASW03 and ASW13 which had a score of 19.30 and 33.35 respectively. This was unexpected, as ASW03 and ASW13 both greatly prevented time-dependent G6PD enzyme activity loss in vitro which indicates enzyme stabilisation. The inhibitors ASW22, ASW23, ASW24, ASW25, ASW26, ASW27 and ASW28 also scored well for binding affinity to the NADP+ binding sites, with the exception of ASW25 which did not score as well for the binding to the catalytic NADP+ site. When trialled against recombinant enzyme, ASW25 exhibited great inhibition of enzyme activity and thus this weakened binding affinity to the catalytic site may be of interest.

The docking simulations also presented predictions of ligand-enzyme interactions which were obtained following analyses of the docking conformations, with the 2D interaction map of the drug to amino acids of the enzyme region presented for each drug. Amino acids within the structural NADP+ binding region that have interactions with the trialled small molecules are summarized in Table 3.4.2.2, with the 2D interaction maps of enzyme-stabilising ASW compounds and associated amino acids illustrated in Figure 3.4.2.1.

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Figure 3.4.2.1 Ligand-enzyme 2D interaction map with G6PD enzyme stabilising ASW compounds. ASW compounds which preserves G6PD enzyme activity were computationally docked into the structural NADP+ substrate binding region of G6PD monomer 1QKI. Ligand-enzyme 2D interaction maps of best conformation were chosen based on cluster size, rank and Goldscore fitness. Abbreviations: G6PD, Glucose-6-phosphate dehydrogenase; 2D, 2-dimensional.

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Table 3.4.2.2 ASW compound interaction with amino acids of G6PD enzyme NADP+ structural binding region determined by computational docking simulations.

Receptor-ligand docking simulations were conducted using Discovery studio 4.1, with the results of the best ligand docking conformation used for amino acid interaction analyses. Drugs were docked into the structural NADP+ binding site of the G6PD enzyme with the 1QKI structure of G6PD used for analysis. The presence of an interaction is nominated with the symbol “Y”. 123

There were several amino acids which had predicted interactions with all the ASW small molecules isolated from the G6PD HTS screen and the substrate NADP+. These were Arg357, Lys366, Arg370, Arg487, Phe501 and Thy503. Phe501 of G6PD was predicted to be a common site of interaction amongst all small molecules but had no predicted interactions with the NADP+ substrate. This residue could therefore contribute to the effect of these small molecules on the enzyme. Nearly every screen-derived small molecule which resulted in increased G6PD enzyme activity via stabilising the enzyme, those being ASW03, ASW04, ASW06, ASW07, ASW08, ASW13 and ASW22 as described in Figure 3.4.1.3 exhibited interaction with Lys497 within the NADP+ structural binding region, with the exception of compound ASW21. Drugs trialled for enzyme stabilising properties which did not prevent enzyme activity loss showed no interaction with this residue, along with inhibitors of the enzyme, with the exception of the compound ASW28. Although ASW28 was isolated from the screen as a G6PD inhibitor, further testing with the small molecule on recombinant enzyme showed little to no changes occurring to enzyme activity following treatment. The software also predicted a lack of interaction with Lys497 by the NADP+ substrate.

Other potential stabilisers which were also predicted to interact with Lys497 included the molecules ASW09, ASW16 and ASW18, which had not been tested on recombinant G6PD enzyme in vitro for enzyme stabilisation due to the unavailability of these compounds. As this residue was a common amino acid interaction amongst G6PD stabilisers, these compounds may be worth pursuing as they may also share enzyme stabilising qualities. However, as seen with compound ASW28 which does not alter enzyme activity during treatment, interaction with the residue does not always result in the preservation of enzyme activity. Further exploration would be necessary to understand the importance of Lys497 to the stability of the G6PD enzyme. ASW06 and ASW07, which are almost identical in molecular structure except for a single molecular change, were predicted to interact with all the same amino acids within the structural NADP+ binding site with only one additional amino acid interaction observed with the compound ASW07 with Glu494.

The compounds ASW03, ASW06, ASW07 and ASW22 were also studied for changes that occurred to G6PD enzyme kinetics when cultured in the presence of the ASW compounds under standard G6PD enzyme assay conditions. Changes to enzyme kinetics were assessed by varying a single substrate concentration whilst keeping the other substrate concentration constant and at saturation, with the enzyme reaction being measured immediately following the addition of the small molecules with no enzyme pretreatment period.

Treatment with ASW03 did not affect the Km of the G6PD enzyme for either substrates, but raised the maximum velocity of the G6PD enzyme for substrate NADP+ from 0.523 ± 0.011 AU/hour under control conditions to 0.612 ± 0.015 AU/hour (Figure

3.4.2.2.B) with ASW03. No significant changes occurred to the enzyme’s Vmax for substrate G6P (Figure 3.4.2.2.C). Treatment with ASW06 did not alter the enzyme Km + for either substrates or the Vmax for NADP but significantly decreased the Vmax for substrate G6P to 0.357 ± 0.007 AU/hour from the normal maximum velocity of 0.420 ± 0.012 AU/hour under control conditions (Figure 3.4.2.2.C). ASW22 treatment resulted + in a significant decrease in the Vmax of both NADP and G6P to 0.466 ± 0.010 AU/hour and 0.359 ± 0.013 AU/hour respectively (Figure 3.4.2.2.C). No changes to enzyme Km for either substrates occurred when G6PD was cultured in the presence of the drug ASW22.

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Figure 3.4.2.2 Effect of G6PD-stabilising ASW small molecules on G6PD enzyme kinetics. (A) Michaelis-Menten kinetics of recombinant human wild-type G6PD enzyme treated with ASW enzyme stabilsers. (B) ASW small molecule treatment effect on the Km of G6PD enzyme. There were no significant + changes occurring to the Km of substrate NADP or G6P. (C) ASW G6PD stabilser effects on Vmax of G6PD enzyme. Treatment with compound ASW03 significantly increased the enzyme maximum velocity for

+ + substrate NADP to 0.612 ± 0.015 AU/hour (p = 0.0002) compared to the control Vmax of NADP of 0.523 + ± 0.011 AU/hour. ASW22 treatment resulted in a significant decrease of Vmax for both substrates NADP and G6P to 0.466 ± 0.010 AU/hour (p = 0.0133) and 0.359 ± 0.013 AU/hour (p = 0.0081) respectively.

Compound ASW06 decreased Vmax of G6P from 0.420 ± 0.012 AU/hour to 0.357 ± 0.007 AU/hour (p = 0.0061). Statistical analyses were performed using two-way ANOVA.

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3.4.3 G6PD-stabilising ASW compounds exhibit G6PD variant specificity for action

The small molecules ASW03, ASW06, ASW07 and ASW22 were previously shown in in vitro studies to preserve the activity of wild-type G6PD enzyme (G6PD_WT), which would spontaneously decline at room temperature. The G6PD-stabilising characteristics of these compounds present an opportunity for use in the treatment of G6PD enzyme deficiency, of which single point mutations within the G6PD gene result in amino acid changes leading to increased instability of the enzyme (Beutler & Vulliamy, 2002; Luzzatto, 2006; Naylor et al., 1996; Vulliamy, Mason & Luzzatto, 1992). Here, newly identified G6PD-stabilising ASW small molecules obtained from an in vitro screen against commercially sourced recombinant G6PD wild-type protein were cherry-picked and trialled against two common pathological G6PD deficiency variants, the A-- variant (G6DP_A--) and the Mediterranean variant (G6PD_mediterranean) (Beutler, 1994).

Plasmid encoding wild-type G6PD protein within the pSJ3 vector, received as a gift from Dan Ye from Fudan University, Shanghai, China were used as a template in the construction of the G6PD deficiency variants. Using site-directed mutagenesis, specific base pair changes were introduced into G6PD to mimic the G6PD deficiency variant described in Experimental methods 3.3.3, with mutagenesis validated by sequencing prior to protein expression (Figure 3.4.3.1.B). G6PD protein was expressed containing an eight-histidine residue at the N-terminal end of the protein for aid in protein purification via the use of metal affinity chromatography resin, with a TEV protease cleavage site between the His-tag and the protein for removal of the tag post-purification. The G6PD gene had been inserted after the T7 promoter DNA sequence, with expression of the protein being controlled under the lac operator induced via the addition of IPTG (Figure 3.4.3.1.A). Protein expression was induced at lowered temperature (16 ºC) to reduce protein misfolding, and bacteria were harvested 16-20 hours after induction.

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Figure 3.4.3.1 G6PD mutant variant plasmid generation. (A) Plasmid map of pSJ3 vector used for full- length recombinant G6PD deficiency variant expression in E. coli. The G6PD protein consisted of 8 histidine tag at protein N-terminal with TEV cleavage site for His-tag removal post-purification, with G6PD variants being constucted using site-directed mutagenesis from the pSJ3-His-G6PD_WT template as described in the methods section. Primer sequences and methods used for the construction of G6PD deficiency variants are as described the Experimental methods section 3.3.3. (B) Sequence validation for constructed G6PD deficiency mutants. G6PD deficiency variants G6PD_A-- and G6PD_Mediterranean were constructed as described in the methods section, with success of site-directed mutagenesis validated by DNA sequencing of the constucted plasmids.

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pSJ3 plasmid vector containing G6PD_WT (wild-type variant), G6PD_A-- (Class II deficiency variant) or G6PD_Mediterranean (Class I deficiency variant) were expressed in BL21 (DE3) E. coli alongside vectors encoding chaperone proteins DnaK, DnaJ, GroEL/GroES and tRNA for arginine, leucine and isoleucine to improve the solubility of expressed protein (Supplementary 9). Folded active proteins were harvested from the soluble fraction of the separated bacterial lysate and purified as described in Experimental methods 3.3.6, with purified fractions being analysed via SDS-PAGE gel for the assessment of protein purity. All three G6PD variants, G6PD_WT, G6PD_A-- and G6PD_Mediterranean were of acceptable purity, with no additional bands observed outside of the 50– 75 kDa band range (Figure 3.4.3.2.A). Faint bands were detected at 75 kDa and just below the band of interest, with these contaminating bands determined to be chaperone proteins GroEL/GroES as determined by mass spectrophotometry on the extracted protein band (Supplementary 10).

The protein fractions were validated to be G6PD protein by western blotting using primary antibody for G6PD protein (Sigma: HPA000247) (Figure 3.4.3.2.B), and protein samples were dialyzed and concentrated for buffer exchange prior to measurement of G6PD enzyme activity. Purified recombinant wild-type G6PD exhibited an activity of 0.798 ± 0.018 AU/hour/µg enzyme, with G6PD deficiency variant A-- having mildly reduced activity of 0.677 ± 0.007 AU/hour/µg and the Mediterranean variant exhibiting 0.208 ± 0.010 AU/hour/µg activity (Figure 3.4.3.2.C). Thus, the isolated G6PD_A-- and G6PD_Mediterranean protein were of 84.84% and 26.07% enzyme activity compared to the wild-type protein respectively.

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Figure 3.4.3.2 SDS-PAGE analysis of purified G6PD deficiency variants with measurement of G6PD enzyme activity. (A) Coomassie stained SDS-PAGE gel of purified G6PD variants showing purified fractions of G6PD_WT (Lane 1), G6PD_A-- (Lane 2) and G6PD_Mediterranean (Lane 3). Lane M consisted of SDS-PAGE gel markers from Bio-RAD Percision Plus ProteinTM KaleidoscopeTM Prestained protein. (B) Western blot of purified recombinant G6PD protein consisting of G6PD_WT (Lane 1), G6PD_A-- (Lane 2) and G6PD_Mediterranean (Lane 3). (C) G6PD enzyme activity of purified recombinant G6PD fractions normalized to protein concentration as determined by Bradford assay. The activity of purified G6PD_WT was measured to be 0.798 ± 0.018 AU/hour/µg, with G6PD_A-- having a decreased activity of 0.677 ± 0.007 AU/hour/µg and G6PD_Mediterranean activity at 0.208 ± 0.010 AU/hour/µg.

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Isolated recombinant G6PD enzymes of the wild-type, A-- and Mediterranean variants harvested and purified from the bacterial expression system were subjected to small molecule testing for improvements to enzyme stability in the presence of the lead G6PD-stabilisers ASW03, ASW06, ASW07 and ASW22. The G6PD enzyme activity assays were conducted under the same experimental conditions as previous G6PD enzyme assays, with freshly purified G6PD protein stored in Tris-buffer containing 20% glycerol being used in place of the commercially sourced fractions. G6PD protein was maintained in the presence of the ASW small molecules with enzyme activity being measured at time 0 directly after drug administration and at time 30 minutes, whereby the enzyme had been incubated in the presence of the drugs for 30 minutes to determine the enzyme stabilisation capacity of the drugs.

All G6PD protein variants exhibited significant loss of enzyme activity following the 30 minutes incubation period, with G6PD_WT exhibiting a loss of enzyme activity from 0.959 ± 0.029 AU/hour/µg to 0.5376 ± 0.019 AU/hour/µg over the 30 minutes incubation period, which was a 43.95% activity loss. The G6PD_A-- had an initial activity of 0.891 ± 0.007 AU/hour/µg enzyme (Figure 3.4.3.3.A), which was 92.89% the enzyme activity of the wild-type enzyme variant. Following the incubation period, the G6PD_Mediterranean variant activity reduced to 0.671 ± 0.031 AU/hour/µg, 24.74% activity compared to the wild-type (Figure 3.4.3.3.A). Purified G6PD_Mediterranean had the lowest enzyme activity of the three purified G6PD fractions, exhibiting an enzyme activity of 0.278 ± 0.011 AU/hour/µg, 29.99% of that of the wild-type variant. The enzyme also experienced a mild loss of activity, with the incubation resulting in a loss of enzyme activity by 26.73% to 0.204 ± 0.006 AU/hour/µg (Figure 3.4.3.3.A). The G6PD- stabilising ASW small molecules trialled in this experiment prevented G6PD_WT enzyme activity loss, with compound ASW06 exhibiting greatest activity maintenance (+40.76% to control, 0.757 ± 0.008 AU/hour/µg, p = 0.0001), followed by small molecule ASW03 (+26.64% to control, 0.538 ± 0.018 AU/hour/µg p = 0.0004), ASW22 (+15.07% to control, 0.619 ± 0.069 AU/hour/µg, p = 0.0406) and ASW07 (+14.60% to control, 0.616 ± 0.044 AU/hour/µg, p = 0.0485) (Figure 3.4.3.3.B).

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Figure 3.4.3.3 G6PD-stabilising small molecules exhibit G6PD variant specificity. (A) Loss of enzyme activity for purified recombinant G6PD enzyme fractions following 30 minutes enzyme incubation. All three enzymes G6PD_WT, G6PD_A-- and G6PD_Mediterranean exhibited a loss of enzyme activity following incubation. (B) Effect of ASW small molecules on G6PD_WT, (C) G6PD_A-- and (D) G6PD_Mediterranean enzyme stability. Each reaction was performed in triplicate, represented as mean absorbance with standard deviation. Statistical analyses were performed 2-way ANOVA.

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Small molecules ASW03 and ASW06 also mildly, but significantly increased G6PD_WT enzyme activity prior to time-dependent activity loss, with ASW03 increasing activity by 10.94% (1.064 ± 0.019 AU/hour/µg, p = 0.007), and ASW06 increasing activity by 8.12% (1.037 ± 0.008 AU/hour/µg, p = 0.049) (Figure 3.4.3.3.B). Similarly, these two small molecules also increased the enzyme activity of variant G6PD_A-- before the enzyme-drug incubation period, with ASW03 resulting in 1.037 ± 0.028 AU/hour/µg (+16.40%, p = 0.0028) and ASW06 exhibiting enzyme activity of 1.019 ± 0.019 AU/hour/µg (+14.38%, p = 0.0087) prior to G6PD_A-- activity loss (Figure 3.4.3.3.C). Compound ASW03 did not however result in significant preservation of the enzyme’s activity following the 30 minutes incubation period, with no significant changes to enzyme activity observed when compared to the control group. Compound ASW06 and ASW07 significantly improved enzyme activity post-incubation, with ASW06 treatment resulting in 0.787 ± 0.055 AU/hour/µg (+17.33%, p = 0.017) and ASW07 preserving activity to 0.847 ± 0.020 AU/hour/µg (+26.35, p = 0.0004) (Figure 3.4.3.3.C). The G6PD-stabiliser ASW22, which prevented activity loss of the G6PD_WT protein did not significantly affect enzyme activity of the G6PD_A-- variant (Figure 3.4.3.3.C).

Purification of G6PD_Mediterranean under identical conditions resulted in a protein batch with reduced activity of 0.671 ± 0.031 AU/hour/µg, 24.74% the activity of the wild-type counterpart (Figure 3.4.3.3.A). Treatment with ASW03 significantly increased enzyme activity by 11.62% (0.310 ± 0.008 AU/hour/µg, p = 0.044) compared to enzyme treated with DMSO control prior to time-dependent activity loss, with other small molecules not significantly affecting enzyme activity. Following the incubation period, no G6PD-stabilising effects were observed by the ASW small molecules except molecule ASW06, which mildly prevented enzyme activity loss resulting in activity of 0.255 ± 0.016 AU/hour/µg, +25.04% above that of the control (p = 0.0014) (Figure 3.4.3.3.D). Therefore, ASW03, ASW06, ASW07 and ASW22 stabilised G6PD_WT, but only ASW06 and ASW07 were effective in stabilising the G6PD_A-- variant. ASW06 mildly prevented a loss in activity of the G6PD_Mediterranean enzyme, for which the other drugs were ineffective. Compound ASW03 increased the activity of all three enzyme variants, with the increase in activity being observed prior to the time-dependent activity loss, whilst ASW06 functioned similarly on both the wild-type and A-- protein, but not on the Mediterranean variant.

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3.5 Discussion

ASW small molecules stabilises G6PD enzyme to maintain activity

The lead small molecules identified from the G6PD activator high-throughput screen were purchased from various drug distributors, with drugs purchased from the same suppliers which sourced the small molecules used in the initial screen where possible. Of the 28 lead compounds identified from the initial screen, 20 were readily available and were procured for further studies. These compounds were subjected to re- testing of the G6PD enzyme activity assay to validate the effects observed on the enzyme and to ensure the same drugs used in the screen were obtained, with the experiments performed manually rather than under automated conditions due to the absence of these instruments.

The G6PD enzyme activity assays were repeated under the same parameters used in the initial screen, that is, with the same 15 minutes small molecule-enzyme pretreatment period. A similar experiment was also conducted alongside this assessment, with a reduced drug-enzyme pre-treatment time of 5 minutes. This was to determine the importance of this drug-enzyme interaction time, as well as to assess if the drugs were able to cause changes to enzyme activity in absence of pre-treatment. The G6PD inhibitor DHEA, which served as a negative control for this experiment significantly reduced enzyme activity following both enzyme pre-treatment durations, with enzyme activity being reduced by 13.25% following the 5 minute incubation period with the inhibition being increased to 45.42% following 15 minutes incubation (Figure 3.4.1.1). Only two of the trialled ASW compounds, ASW13 and ASW25, altered G6PD enzyme activity following the shorter incubation period, whilst ten out of twenty ASW compounds significantly altered G6PD enzyme activity following the longer incubation period of 15 minutes enzyme pre-treatment. These compounds were ASW03, ASW04, ASW06, ASW07, ASW08, ASW13, ASW21, ASW22, ASW24 and ASW25 (Figure 3.4.1.1.B). Thus, the efficacy of the drugs in altering G6PD enzyme activity was determined to be dependent on the time length of enzyme-drug pre-treatment, with a larger incubation time resulting in increased enzyme activity.

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The 47.99% decrease in mean G6PD activity between the solvent control groups with different drug-enzyme incubation periods (Figure 3.4.1.1.A and B) suggests time- dependent loss of enzyme activity occurring in vitro. This was of interest as it suggested instability of the G6PD enzyme during the experimental procedure, with ASW compound treatments preventing the time-dependent loss of enzyme activity instead of increasing the enzyme specific activity. This loss of enzyme activity with time was confirmed with the time-dependent incubation assay, whereby varying lengths of enzyme incubations were compared and an inverse relationship between enzyme incubation timelength and enzyme activity was observed (Figure 3.4.1.2). Increasing the enzyme incubation time resulted in decreased enzyme activity, indicating enzyme instability or degradation with time. This is not uncommon, as there are enzymes known to denature slowly even at physiological temperature of 37 ºC which may be due to contaminating proteases or the oxidative process. The conditions within the cell often prevents this degradation, with the high protein concentration serving as a stabiliser to improve enzyme half-life (Hinkson & Elias, 2011).

As purified recombinant protein was used in these assays, the gradual loss of activity resulting from destabilisation of the enzyme could have been caused by a lack of stabilisers within the mixture. The presence of several of the ASW compounds, which were ASW03, ASW04, ASW06, ASW07, ASW08, ASW13, ASW21 and ASW22 maintained the enzyme activity during this period. These results suggested these small molecules behaved as G6PD stabilisers, maintaining enzyme activity rather than activating the enzyme similar in function as the AG1 small molecule recently identified by the Mochly-Rosen lab (Hwang et al., 2018). Following these results, the subsequent G6PD enzyme activity assays were modified with an increased enzyme pre-treatment time-length of 30 minutes to allow for enzyme activity loss to occur with enzyme activity being measured at the start and end of the pre-treatment period to assess for capabilities of the small molecules to stabilise and maintain enzyme activity.

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Under at least three independent experiments performed with identical conditions and handling, several of the small molecule G6PD stabilisers were found to effectively maintain enzyme activity and prevent a loss of activity. These were ASW03, ASW13, ASW21 and ASW22, which greatly preserved enzyme activity when cultured with the enzyme. Compared to the control group which exhibited enzyme destabilisation resulting in 63.25% remaining activity following incubation in vitro, these compounds maintained G6PD enzyme activity with up to 92.38% activity being preserved (Figure 3.4.1.3). The only other known molecule which stabilises the G6PD enzyme, AG1, was reported to increase wild-type recombinant G6PD activity by 20% when cultured in vitro (Hwang et al., 2018). However, the length of enzyme pre-treatment which dictates the extent of enzyme denaturation and activity loss may vary from our methods and thus assessing drug efficacies between the two studies will not provide an accurate comparison. Other predicted stabilisers including ASW04, ASW06, ASW07 and ASW08 also showed a trend of increased G6PD activity following incubation, however this was below the threshold of statistical significance. This could be due to molecules being trialled at a single fixed concentration of 10 µM, which may not be a sufficient dose for enzyme stabilisation.

Compounds ASW24 and ASW25 were previously shown to reduce enzyme activity decreased G6PD enzyme activity in both timepoints, that is at the start of the reaction and 30 minutes after, with only compound ASW25 exhibiting a statistically significant decrease in enzyme activity without the need for enzyme pre-treatment. These results suggest that the inhibitors functions instantly and without the need for preincubation with the enzyme to exhibit this effect, possibly by means of competition with the enzyme’s substrates, or may be due to destabilisation of the enzyme. Previously identified inhibitors of G6PD, such as gallated catechins were found to be competitive inhibitors, competing with substrate NADP+ resulting in enzyme inhibition (Shin et al., 2008). Further exploration will be required to understand how ASW inhibitors function to reduce enzyme activity. ASW01, ASW02, ASW12, ASW15, ASW19, ASW20, ASW23, ASW26, ASW27 and ASW28 did not significantly alter G6PD enzyme activity, and were de-prioritised for future experiments due to the need for lead specialisation (Supplementary 3). The lack of effect seen with treatment of these small molecules which were isolated from the G6PD HTS as activators of the G6PD enzyme could be due

136 to inconsistent source of the drugs from the supplier, difference in purity of the acquired materials or loss of drug potency during transit of the molecule.

Enzyme stabilisation of ASW small molecules occurs with interaction of the structural NADP+ binding site of the G6PD enzyme

Computational molecular docking simulations were used to predict G6PD binding affinity and drug-enzyme interactions of the G6PD HTS-derived ASW small molecules. The NADP+ binding regions on the G6PD enzyme were the focus of this study, with docking simulations directed at both the catalytic and structural NADP+ binding domains of the enzyme as small molecules tend to interact with substrate binding sites of enzymes. Furthermore, several ASW compounds were found to stabilise the G6PD enzyme, preventing a loss in enzyme activity upon further testing. As the structural NADP+ binding site of the G6PD enzyme is involved in enzyme stabilisation (Wang, Chan, Lam & Engel, 2008b), this region was predicted to be the target of the G6PD-stabilising ASW small molecules, and was therefore a focus for the molecular docking studies. Specific negative controls could not be issued within the receptor-ligand docking simulations as interactions could occur regardless of the specificity of the molecules which will provide a binding affinity score. However, a positive control, which in our studies was the natural substrate NADP+, could be used to generate a benchmark score for complete interaction of the ligand with the enzyme.

There were no correlation between the improvement of enzyme activity and the binding affinity of the drug to NADP+ binding regions, which was not unexpected as several of the small molecules which had been shown to increase enzyme activity could not be validated when the experiment was repeated in-house. There is the possibility that the results obtained from the G6PD HTS for these small molecules were false positives or could be due to the purchased drugs not being of the same potency as the drugs used in the screen. The computational docking simulations indicated compound ASW18 to have the best binding affinity of the screen-derived small molecules for the structural NADP+ binding region of the G6PD enzyme, which is involved in the maintenance of long-term enzyme stability (Wang, Chan, Lam & Engel, 2008a). This drug however had not been trialled further past the G6PD HTS due to drug unavailability by compound suppliers. This small molecule may be worth pursuing in the future, as the results from 137 computational analyses suggest a good interaction with the structural NADP+ binding region which may attribute to the drug being able to stabilise the G6PD enzyme.

ASW12 did not prevent time-dependent activity loss of G6PD enzyme when trialled in vitro on recombinant G6PD despite having scored highest in binding affinity for the NADP+ catalytic region with decent binding affinity for the structural binding region. This discrepancy could be due to instability of the drug which could have lost potency during transit and delivery which had occurred at room temperature, or the drug being a false positive within the computational analyses. The lead G6PD stabilising small molecule ASW03 which exhibited consistent and robust enzyme activity preservation also exhibited low computational binding affinity towards the structural NADP+ binding site of the G6PD enzyme, which is involved with stabilising the enzyme (Wang, Chan, Lam & Engel, 2008b). This could suggest a transient drug-enzyme interaction, or that the drug interacts with a different region of the enzyme to improve stability. Further experiments will be needed to better understand how ASW03 stabilises the G6PD enzyme. The poor drug-enzyme Goldscore fitness, suggesting weak binding affinity, does not always indicate a lack of interaction of a drug to the assessed region of the enzyme, as molecules could interact with a particular binding zone transiently eliciting a change in the enzyme conformation.

ASW compounds that faithfully preserve and maintain G6PD enzyme activity in vitro were the small molecules ASW03, ASW04, ASW06, ASW07, ASW08, ASW13 and ASW22. These compounds likely stabilise the enzyme and prevent a loss of activity by enzyme degradation through predicted interactions with a single amino acid, Lys497. This residue is located within the structural NADP+ binding region, with no computational predicted interactions observed with other ASW small molecules which did not exhibit this stabilisation effect. Thus, this lysine residue may play an important role in stabilising the G6PD enzyme via pharmacological means and could be used as a target to identify other enzyme-stabilising small molecules or for modifications to be made to foster interactions with this residue.

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Several of the small molecules ASW03, ASW06, ASW07 and ASW22 which consistently stabilised the G6PD enzyme preventing enzyme activity loss also altered enzyme kinetics. No changes occurred to the Km of the enzyme for either substrates NADP+ or G6P, but ASW03 increased the enzyme maximum velocity in the presence of substrate NADP+, suggesting the increase in enzyme activity may not be solely due to the enzyme stabilisation. Treatment with ASW06 decreased the maximum velocity of the enzyme for substrate G6P, whilst the nearly identical molecule ASW07 did not cause any changes to the G6PD enzyme kinetics. Both molecules stabilise G6PD enzyme when trialled in vitro, with the enzyme kinetics results suggesting no further activation of the enzyme, but both molecules may be different in efficacy due to a single change in molecular structure which may be relevant to future medicinal chemistry studies. Treatment with ASW22 decreased maximum enzyme velocities for both NADP+ and G6P with no changes observed to substrate Km, suggesting the increase in enzyme activity observed following enzyme incubation with ASW22 likely to be caused by prevention of time-dependent activity loss rather than modulation of enzyme activity.

Surface Plasmon Resonance (SPR) of the three leading G6PD stabilisers ASW03, ASW08 and ASW22 to recombinant human G6PD enzyme further validated small molecule-enzyme interaction and binding, with binding affinity reported as the equilibrium dissociation constant (KD) values of 18 ± 6 µM, 40 ± 15 µM and 12 ± 7 µM respectively (Supplementary 11). These KD coefficients were in range and in good accordance supporting receptor-ligand binding (Du, Zhang, Luo, Chen, Shen & Jiang,

2006), with the values also being in good agreement to the small molecule IC50 values (Supplementary 4, 5 and 7). SPR testing of these small molecules are preliminary results of an ongoing project and were conducted by Lorna Wilkinson-White at the Sydney Analytical Fragment Based Drug Discovery Facility of the University of Sydney, Australia. These results support the interaction of the small molecules with the G6PD enzyme and suggests improved dimerization of the enzyme following compound treatment.

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The G6PD-stabilising ASW small molecules exhibit G6PD variant specificity for enzyme stabilisation

Most G6PD enzyme deficiency variants differ from the wild-type G6PD by a single amino acid change, with more than 140 G6PD variants having been described (Beutler, 1994; Cappellini & Fiorelli, 2008). As the G6PD-stabilisers were predicted to bind and interact with wild-type G6PD enzyme, specifically the structural NADP+ binding region as described in the earlier sections of this chapter, proven G6PD-stabilising molecules were trialled on purified recombinant G6PD variants to determine if there were key amino acids involved with the stabilisation of the enzyme. The G6PD variants were over-expressed in E. coli with plasmid encoding the wild-type G6PD, or G6PD deficiency variants constructed using the wild-type plasmid as a template via site-directed mutagenesis as described in Experimental methods section 3.3.3.

Over-expression of the wild-type G6PD enzyme via the pSJ3 plasmid vector in BL21 (DE3) cells resulted in the G6PD protein being expressed in an aggregated state, with most of the protein presenting within the insoluble fraction of the lysate and only a miniscule amount of the protein being detected within the soluble fraction of the lysate. Protein aggregation and misfolding is a common complication that occurs during recombinant protein overproduction in cells (King & Betts, 1999; Schrödel & de Marco, 2005) and often the misfolded protein trapped in the inclusion bodies within cells are in dynamic equilibrium with their soluble fraction counterparts (Carrio & Villaverde, 2001). Therefore, plasmids encoding chaperone proteins GroEL/GroES, DnaK, DnaJ and tRNAs for amino acids Arginine, Lysine and Isoleucine were co-expressed with all G6PD protein expression to improve protein solubility and increase the yield of functional G6PD protein (Supplementary 9), which were then harvested and subjected to purification.

Two common G6PD deficiency variants, the A-- and Mediterranean variant, were selected for protein over-expression in bacteria, purification and small molecule trial to assess for G6PD variant specificity by the G6PD stabilising ASW drugs. The G6PD A-- variant of deficiency Class III is believed to be caused by the decreased folding of the enzyme caused by a double mutation not affecting the active site of the enzyme but ultimately causing the formation protein with decreased intracellular stability (Gomez- Gallego, Garrido-Pertierra & Bautista, 2000; Town, Bautista, Mason & Luzzatto, 1992), whilst the effect of mutations in the Mediterranean mutation of Class II deficiency

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(Beutler, 1994) is left to be elucidated. Protein purification of the harvested G6PD over- expressing bacterial lysate via metal affinity chromatography resulted in purified G6PD protein fractions of decent purity, however with two faint contaminating bands located in regions above the G6PD protein band (~75 kDa) and directly below (~55 kDa). These contaminating bands were identified to likely be of the co-expressed chaperonin protein GroEL/GroES as indicated by mass spectrophotometry of the excised bands (Supplementary 10), with the protein functioning as a tetradecameric subunit structure consisting of fourteen identical 58 kDa monomers for assistance of protein folding (Purich, 2010).

Due to the unstable nature of the purified G6PD deficiency mutants which cause rapid enzyme activity loss after the purification process, the removal of the N-terminal His-tag via TEV protease cleavage and secondary metal affinity chromatography were omitted, reducing the time interval from when the protein undergoes purification, and when G6PD enzyme activity were measured and compared to determine the small molecule interaction with the purified protein. The purified recombinant protein exhibited typical enzyme activity, with the deficiency variants exhibiting reduced enzyme activity compared to the purified wild-type G6PD. The A-- G6PD variant had 84.84% normal enzyme activity, which was slightly elevated than the activity reported in the literature (Town, Bautista, Mason & Luzzatto, 1992), but not uncharacteristic of a Class III G6PD deficiency variant of which typically exhibits 10 – 60% normal enzyme activity (WHO, 1989). Isolated G6PD Mediterranean variant exhibited 26.07% enzyme activity, also of above average activity for Class II deficiency variants which typically express 1 – 10% enzyme activity (WHO, 1989). The mild increase in enzyme activity observed with the two purified G6PD deficiency variants compared to the wild-type counterpart could be due to contaminating chaperone proteins, which may have increased stability or activity of the mutant or may have affected the activity of the wild-type protein.

Similar to the recombinant human wild-type G6PD enzyme used in the G6PD HTS and preliminary assays, which were commercially purchased, all three purified recombinant G6PD protein variants exhibited enzyme activity loss with time when cultured in vitro. G6PD-stabilising ASW small molecules were administered to the purified recombinant protein and allowed to incubate for 30 minutes before measuring G6PD enzyme activity to assess for G6PD-stabilising capacity of the molecules and to assess for G6PD variant specificity. Compound ASW06 was most robust, preventing a 141 loss in enzyme activity in all three protein variants, whilst also mildly increasing enzyme activity of the wild-type and A-- variants prior to the incubation period, suggesting intrinsic increase to enzyme activity. The G6PD-stabiliser ASW03 reduced activity loss of only the wild-type G6PD, with no prevention of activity loss observed in either of the two deficiency variants exhibiting variant bias. This observation is unlike the known G6PD-stabilising molecule AG1 identified by Hwang et. al, which improved the stability of both the wild-type and Canton deficiency enzyme variant (Hwang et al., 2018). A mild increase in activity was observed with ASW03 treatment in all enzyme variants prior to enzyme activity loss, indicating enzyme activity modulation rather than preservation of + enzyme activity, consistent with the observed improved Vmax for substrate NADP following treatment, as was determined by enzyme kinetics analyses. ASW07, which is nearly identical to ASW06 except for a single molecule change exhibited enzyme- stabilising effects on the wild-type and A-- variant, but not in the Mediterranean G6PD variant, suggesting that small changes to molecular structure may impact on binding affinity for G6PD enzyme, and that the mutation present in the Mediterranean variant may also hinder the binding of molecule ASW07. Similar results were also observed with ASW22 treatment, with both molecules ASW07 and ASW22 also lacking enzyme activating effects on the tested enzymes.

In conclusion, the G6PD-stabilising ASW small molecules do exhibit G6PD variant specificity, with several molecules functioning more effectively on certain enzyme variants, whilst having no effect on others. With more than 140 G6PD variants having been described and the majority of the mutations causing single amino acid changes (Beutler, 1994; Cappellini & Fiorelli, 2008), these G6PD-stabilisers should be trialled on other enzyme variants to better elucidate crucial enzyme features and mutations which may be responsible for the maintenance of enzyme stability, or the increase of enzyme activity, and may be useful in molecule optimization to improve the efficacy and binding of the ASW small molecule stabilisers.

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Chapter 4 Effect of novel G6PD-stabilisers on health and lifespan

Effect of novel G6PD-stabilisers on health and lifespan

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4.1 Introduction

Biological ageing is an important risk factor to disease and is accompanied by many characteristics such as a functional decline in physical performance and frailty, often impacting on quality of life. To address the question of whether these G6PD- activators, now shown to function as enzyme stabilisers, could improve healthspan and increase lifespan in vivo, we utilized the nematode model organism Caenorhabditis elegans. C. elegans are a popular model for studying ageing and longevity due to their relatively short lifespan, rapid generation time, cost-effectiveness with maintenance, and the well-defined genetic and environmental factors that affect their lifespan (Blagosklonny, Campisi & Sinclair, 2009; Braeckman & Vanfleteren, 2007; Fontana, Partridge & Longo, 2010; Guarente & Kenyon, 2000). Furthermore, the lack of further cell division or regeneration of cells in fully grown C. elegans makes the nematode an excellent model organism to study ageing.

The primary goal of this field has been to improve late-life health and alleviate the increased risk of disease with advanced age, to maintain quality of life. In order to assess whether these compounds have a functional impact on ageing, the effect of these newly discovered G6PD-stabilising compounds on healthspan needs to be assessed through measuring physical performance. C. elegans is a useful tool to not only assess lifespan due to the nematode’s relatively short lifespan of three weeks, but also allows for changes in physical activity and performance to be compared. It is unlikely that changes in lifespan measured in this model will translate directly into changes in human lifespan, but there is experimental evidence suggesting interventions that extend lifespan in C. elegans worms are effective in treating age-related diseases such as cancer and neurodegenerative diseases (Collins, Evason & Kornfeld, 2006; Petrascheck, Ye & Buck, 2007).

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Figure 4.1 The G6PD enzyme sequence is highly conserved across evolution. Amino acid alignment of G6PD homologues in humans (G6PD_HUMAN), mouse (G6PD1_MOUSE), fruit fly (G6PD_DROME), yeast (G6PD_YEAST), and nematodes (G6PD_CAEEL). Asterisk (*) denotes fully conserved resides, colon (:) represents strongly similar properties and period (.) represents weakly similar properties. (A) residues 198 - 205 and (B) residues 42 - 48 is completely conserved between all species.

In order to assess whether the small molecule G6PD-stabilising molecules discovered in this project were effective in vivo, and to assess the initial hypothesis that small molecules which increased G6PD activity would be effective in maintaining health during biological age, in this chapter we tested the effects of the newly discovered G6PD- stabilisers on health and lifespan in C. elegans. The protein sequence for the NADP+ binding region of the G6PD enzyme is highly conserved across evolution, including between humans, mice, Drosophila, yeast and C. elegans with the G6P binding domain RIDHYLG/K (residues 198 - 205; single-letter amino acid code) (Figure 4.1.A) and the dinucleotide-binding region GASGDLA (residues 42 - 48) (Figure 4.1.B) which is believed to be important for NADP+ binding being completely reserved (Bautista, Mason & Luzzatto, 1995; Kletzien, Harris & Foellmi, 1994). Using receptor-ligand docking simulations as mentioned in Chapter 3, we had predicted that the G6PD-stabiliser small molecules discovered here might interact with the NADP+ substrate binding region of the G6PD enzyme to stabilise and preserve enzyme activity. Therefore, changes observed in C. elegans health during treatment with newly discovered G6PD small molecule stabilisers could be an important proof of principle to determine whether these compounds could potentially be used to improve human healthspan.

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4.2 Materials

4.2.1 Materials and reagents

Materials

Di-Potassium Hydrogen Orthophosphate [AJA2221], Potassium dihydrogen phosphate [Merck Millipore: 104873], Calcium chloride dihydrate [AJA127], Magnesium sulfate hydrated [AJA302], Sodium chloride [AJA465], Peptone [Amyl Media: RM263], Agar [VWR: 20767.298], Cholesterol [Sigma: C3045], 4% Bleach, β-Nicotinamide adenine dinucleotide phosphate hydrate [Sigma: N5755], D-Glucose 6-phosphate disodium salt hydrate [Sigma: G7250], β-Nicotinamide adenine dinucleotide 2′-phosphate reduced tetrasodium salt hydrate [Sigma: N7505], Tris (Hydromethyl Methylamine) [AJAX- 2311], Trizma® hydrochloride [Sigma: T5941], 6-Phosphogluconic acid trisodium salt [Sigma: P7877], trans-dehydroandrosterone [Sigma: D4000], Triton X-100 [Sigma: T8532], Magnesium sulfate [AJAX-302], cOmplete EDTA-free Protease inhibitor cocktail tablets [Roche: 05056489001], Paraquat dichloride [Sigma: 36541], Dimethyl sulfoxide [Sigma: D5879], Magnesium chloride [Sigma: M8266], Ethylenediaminetetra- acetic acid [AJA180], 2-Mercaptoethanol [Sigma: M3701], Diamide [Sigma: D3648],

FeSO4•7H2O [Sigma: 215422], MnCl2•4H2O [Sigma: M3634], ZnSO4•7H2O [Sigma: Z0251], Potassium citrate [Sigma: P1722]

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Reagents

M9 Buffer [42 mM Na2HPO4, 22 mM KH2PO4, 86 mM NaCl, 1 mM MgSO4, add deionized water to 1000 ml, sterilize by autoclaving.] (Stiernagle, 2006)

S Basal Medium [100.1 mM NaCl, 5.74 mM K2HPO4, 44.09 mM KH2PO4, 1 ml cholesterol (5 mg/ml in ethanol), H2O to 1000 ml, sterilize by autoclaving.] (Stiernagle, 2006)

S Complete Medium [1000 ml S Basal medium, 10 mM potassium citrate pH 6.0, 10 ml trace metals solution, 2 mM CaCl2, 3 mM MgSO4, prepared under sterile conditions.] (Stiernagle, 2006)

Trace metals solution [1.86 g disodium EDTA, 0.69 g FeSO4•7H2O, 0.2 g MnCl2•4H2O,

0.29 g ZnSO4•7H2O, 0.025 g CuSO4•5H2O, H2O to 1 litre, sterilized by autoclaving.] (Stiernagle, 2006)

Sodium hypochlorite solution (Bleaching solution) [0.75 ml 5 M NaOH solution, 11.25 ml H2O, 3 ml 4% Bleach.] (Stiernagle, 2006)

Worm lysis buffer [20 mM Tris-HCl, 3 mM MgCl, 1 mM EDTA, 0.1% Triton X-100, 0.02% 2-Mercaptoethanol, Protease inhibitor, pH 7.4]

4.2.2 Instruments

Bacterial incubator (Thermoline, NSW, Australia), Dissecting microscope (Nikon SMZ800, Coherent scientific, NSW, Australia), Refrigerated incubator [1] (Labec, NSW, Australia), Refrigerated incubator [2] (Liebherr, Germany), Shaking incubator (Ratek, Vic, Australia), SpectraMax M Multi-mode microplate reader (Molecular Devices), Branson sonifier 250 (Model 102C, Fisher Scientific)

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4.3 Experimental Methods

4.3.1 Nematode Growth Media (NGM) preparation

Nematode Growth Media (NGM) used for Caenorhabditis elegans growth and maintenance was prepared as previously described (Stiernagle, 2006). 3 g NaCl, 2.5 g

Peptone, 17 g Agar and 975 ml H2O were combined and sterilized by autoclaving. When molten agar was cooled to 55 ºC, 1 ml 1 M CaCl2, 1 ml 1 M MgSO4, 1 ml 5 mg/ml

Cholesterol and 25 ml pH 6.0 KPO4 was added and agar dispensed into petri dishes. Drugs were added to the molten agar and mixed prior to plate preparation to make agar containing drug treatment. The agar plates were allowed to dry overnight before seeding with live OP50 bacteria as food source for C. elegans worms. All reagents used were sterile and agar plate preparations were done aseptically.

4.3.2 Nematode Synchronization

Synchronization via egg lay

Gravid adults were transferred by picking to fresh, seeded NGM plates and allowed to lay eggs for 3 to 5 hours at 20 ºC before being removed. The worms hatched from the remaining eggs on the plate are used as a synchronous cohort.

Synchronization via bleaching

Worms were synchronized by bleaching as described (Stiernagle, 2006). Worms were recovered in a 1.5 ml tube by washing a full plate of worms with M9 buffer. The worms were pelleted by centrifugation for 30 seconds at 800 g at room temperature. The supernatant was discarded, and the pellet washed 3 times to remove remaining bacteria. Bleaching solution was added to the worm pellet, and the mixture agitated by tube inversion for no more than 10 minutes until worms are completely dissolved. Remaining suspended eggs were pelleted by centrifugation and washed 3 times with M9 buffer to neutralize the bleach. The eggs were resuspended in M9 buffer and allowed to hatch overnight at 20 ºC and plated onto fresh OP50 NGM plates the next day.

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4.3.3 Caenorhabditis elegans survival assay

C. elegans wild isolate N2 Bristol strain, obtained from the Caenorhabditis Genetics Centre (CGC) at the University of Minnesota, MN, USA, were cultured and maintained non-starved on NGM agar plates carrying live E. coli OP50 at 20 ºC according to Brenner (1974). All experiments were repeated at least three times, with more than 100 worms used per treatment group. Worms were scored as dead when no response were observed following prodding, and when pharyngeal pumping was no longer present. Worms with internal hatching or had crawled off the NGM media were censored from the study.

To obtain a synchronous cohort, several gravid, egg-laying adult worms were subjected to synchronization via egg laying (Section 4.3.2 Synchronization via egg lay). Synchronized L1-staged larvae were allowed to develop to L4-stage young adulthood. The worms were then transferred onto fresh NGM plates containing 100 µM FuDR to prevent egg laying and vehicle-control or drug at the desired concentration, with the drugs refreshed on Day 5, 10 and 15 of worm adulthood by transferring worms onto freshly made agar via manual hand-picking. Worms were monitored at least once every two days and scored as live or dead. Animal survival was plotted using Kaplan-Meier survival curves and analysed by log-rank test using Graphpad Prism (Graphpad Software, La Jolla California, USA). Survival curves with p values of <0.05 relative to control were considered significantly different.

4.3.4 Antioxidant capacity assessment

Cupric reducing antioxidant power (CUPRAC) was performed as previously described (Apak, Guclu, Ozyurek & Karademir, 2004). Briefly, equal parts of sample, 10 mM

CuCl2, 1 M NH4Ac (pH 7.0), and 7.5 mM Neocuproine were mixed. After 30 minutes, reaction absorbance at 450 nm was measured using a spectrophotometer and recorded against a reagent blank. Results were expressed as µM Trolox equivalent.

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4.3.5 G6PD enzyme activity assay

Sample preparation

C. elegans worms individually hand-picked into tubes containing M9 buffer and were washed three times to remove bacteria. The worms were then resuspended in ice-cold worm lysis buffer containing protease inhibitor and lysed via sonication [10% amplitude, 50% duty cycle, 10 pulses on ice] with complete lysis being confirmed under microscope. The lysate was centrifuged [16’000 g, 10 minutes, 4 ºC] and supernatant collected for G6PD enzyme activity measurement.

G6PD activity measurement

G6PD activity was determined as described previously (Tian et al., 1998). Both 6PGD activity and G6PD+6PGD activity were measured separately, and endogenous G6PD activity was determined by subtracting the 6PGD activity alone from the G6PD+6PGD total enzyme activity value. Each reaction mixture consists of 50 mM Tris-HCl (pH 7.44), 2 mM NADP+ and sample in which G6PD activity was to be measured. Reactions were initiated with the addition of 4 mM G6P + 4 mM 6PGL or 4 mM 6PGL alone and activity was measured using a spectrophotometer at room temperature (24 °C). NADPH production was measured at 340 nm every 30 seconds for at least 20 minutes, and the rate of NADPH determined as a unit of AU/hour.

4.3.6 Caenorhabditis elegans pharyngeal and thrashing rate assays

The physical activity of C. elegans were assessed as previously described, with minor changes (Hahm et al., 2015). Synchronized wild-type L1 larvae were allowed grow to L4 young-adulthood as described in the survival assays. Worms were transferred to treatment plates at the L4 stage of growth, and the contractions of the terminal bulb pharynx were counted for 45 seconds under a dissecting microscope every 2 days (n=6 per treatment group). The movement of the worms were also counted after transfer to S-basal medium for 60 seconds at 20 ºC, with each sigmoidal mid-body bend counted as a single movement.

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4.3.7 Caenorhabditis elegans stress resistance assay

C. elegans resistance to oxidants paraquat and diamide were assessed as previously described (Gusarov et al., 2017). Worms were synchronized via bleaching and allowed to develop and grow on NGM agar at 20 ºC until L4 stage before being transferred to NGM agar containing drugs at 50 µM in the presence of 100 µM FuDR to prevent egg laying and allowed to incubate for 16-20 hours at 20 ºC. The worms were collected into microcentrifuge tubes containing M9 buffer and washed three times to remove bacteria before being transferred into M9 buffer supplemented with oxidants diamide or paraquat at concentration 200 mM and 100 mM respectively and drugs at 50 µM. The tubes were nutated at 20 ºC for 2.5 hours before washing the worms three times with M9 buffer to remove the oxidant prior to transferring to fresh NGM agar containing drug, FuDR and OP50 bacteria. The worms were maintained at 20 ºC and scored daily for survival with survival rate calculated as a ratio of live worms to the total number of worms transferred to the plate after oxidant exposure.

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4.4 Results

4.4.1 Lifespan of C. elegans is improved with ASW compound treatment

The G6PD enzyme stabilisers identified from the high-throughput small molecule screen and investigated in previous chapters were tested for their ability to extend lifespan in the model organism C. elegans. A total of 22 G6PD activators and 8 G6PD inhibitors were identified from the screen, as described in Chapter 2, and several promising candidates were prioritized for further investigation for improvements following treatment in animals. We hypothesised that animals with overall improved G6PD activity, whether by intrinsic enzyme activity or via modulation of enzyme levels, would exhibit extensions in late-life health and overall lifespan. This hypothesis would be in line with previous findings that transgenic G6PD overexpression in wither Drosophila or mice have improved lifespan (Legan et al., 2008; Nobrega-Pereira et al., 2016).

Survival assessments of C. elegans are typically performed on solid medium with live OP50 E. coli bacteria as a food source. However, for ease of experimental protocol, animal handling and to increase the number of drugs able to be tested in a single survival experiment which requires approximately a month to conduct, we decided to conduct a pilot study trialling survival assessment performed using liquid media for C. elegans maintenance. The use of liquid media enabled the nematodes to be maintained in microtiter plates, with the assay having been developed and successfully used previously to screen large libraries of drugs that extend the lifespan of C. elegans worms (Petrascheck, Ye & Buck, 2007; Solis & Petrascheck, 2011). This method, as opposed to the conventional maintenance of C. elegans on solid agar, allowed for the drugs to be readministered without the need for transfer of the worm cohort which has a relatively high loss of animals with each transfer cycle, and for replenishment of a food source when required, which in this case was heat-treated OP50 bacteria.

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A total of 8 different ASW compounds were subjected to further testing for their ability to extend lifespan in C. elegans from a pool of G6PD activators and inhibitors identified from the small molecule screen detailed in Chapter 2, with 7 compounds shown to be G6PD activators functioning via their stabilisation of the G6PD enzyme, which presented a loss of enzyme activity, and 1 compound which was found to inhibit activity of the G6PD enzyme. The G6PD activators assessed were ASW03, ASW06, ASW07, ASW13, ASW15, ASW21 and ASW22, chosen due to their consistency in maintaining recombinant G6PD enzyme activity in vitro as determined upon further exploration described in Chapter 3.

Twenty-one different treatment groups were assessed, including a vehicle control group of DMSO, which was kept consistent amongst all treatment groups at a concentration of 0.2%, with drugs administered as a single dose at the start of nematode adulthood. FuDR was introduced post-development on day 1 of worm adulthood to prevent egg laying for ease of worm maintenance, and worm survival was scored at least once every 2 days until the end of the experiment. At least 200 worms were assessed in each treatment group, with each group occupying 16 separate wells for the duration of the experiment. Worms subjected to treatment with 0.2% DMSO were used as a vehicle control group for this experiment, with treated worms exhibiting a median survival of 13 days, and a maximum lifespan of approximately 38 days, which although long, was not remarkable for worms cultured in liquid media at 20 ºC (Solis & Petrascheck, 2011).

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Figure 4.4.1.1 Effect of G6PD stabilisers on C. elegans survival in a liquid media lifespan assay. ASW compounds were refreshed on Day 5, 10 and 15 of C. elegans worm adulthood. Seven candidate G6PD-stabilisers and one inhibitor were trialled in this pilot study. (A) ASW03 improves the survival of treated worms with concentration 10 µM resulting in a statistically significant improvement, and concentration 50 µM showing a trend in improvement. No change in maximum lifespan was observed. (B) Maximum lifespan was significantly decreased in the presence of ASW06 at both 10 µM and 50 µM concentration. Worms exposed to 10 µM ASW06 had slightly improved survival initially but this observation was not statistically significant. (C) Worm maximum lifespan were shortened significantly at both 10 µM and 50 µM concentrations, but median survival were improved. (D) Both median survival and maximum lifespan of treated worms were significantly impacted at both drug concentrations. (E) No significant changes were observed between drug-treated and control animals. (F) A mild improvement in median survival was observed at concentration 10 µM, without any other significant differences observed. Statistical analyses were determined via Graphpad Prism software using Log-rank (Mantel- Cox) test. Changes resulting from drug treatment are summarized in Table 4.4.1.1.

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Treatments which resulted in significant changes to lifespan are shown in Figure 4.4.1.1, with other treatments available in supplementary [Supplementary 12]. Several G6PD-stabilising compounds, notably ASW03, ASW06, ASW07 and ASW22 exhibited signs of improving the median survival in C. elegans. Worms treated with the compound ASW03 had significantly increased median survival at 10 µM (+23.08%, p = 0.0069), and a trend observed at 50 µM (+23.08%, p = 0.2153), with no significant changes in overall maximum lifespan of the worm when compared to the control with both drug concentrations (Figure 4.4.1.1.A). Treatments with compound ASW06, which shares structural similarity with ASW07 differing only in a single change of a furan to thiophene, did not improve worm median or maximum lifespan but may also be detrimental for worm survival (Figure 4.4.1.1.B), with treatments at both 10 µM and 50 µM reducing lifespan. However, ASW07 improved worm median survival at a concentration of 20 µM (+23.08%, p = 0.0419), with a mild but not significant improvement observed during treatment at a concentration of 10 µM (+38.46%, p = 0.6077). Despite the improvement in median survival following ASW07 supplementation, maximum lifespan in drug- treated worms were still negatively impacted, with worms exposed to both 10 µM and 20 µM concentrations of ASW07 having greatly reduced maximum lifespan (Figure 4.4.1.1.C).

The G6PD-stabilising compound ASW13 negatively affected the survival of C. elegans, with both median survival and maximum survival being reduced at both 10 µM and 50 µM concentrations by 30.77% (50 µM, p < 0.0001) and 40.98% (50 µM, p < 0.0001) respectively (Figure 4.4.1.1.D). Treatment with ASW22 did not significantly affect worm survival, but treatment at 50 µM concentration exhibited a trend in improved median survival (+23.08%, p = 0.0590) and maximum survival marginally (Figure 4.4.1.1.E) and may be worth repeating for validation. The effect of G6PD inhibition was also assessed via the use of the known G6PD inhibitor DHEA (Gordon, Mackow & Levy, 1995) and the G6PD-inhibiting candidate ASW25, which was identified from the small molecule screen. DHEA reduced worm maximum lifespan by up to 23.27% (10 µM, p < 0.0001) with no significant changes to median survival at both 10 µM and 50 µM concentrations (Supplementary 12). In contrast, compound ASW25, which inhibited G6PD activity in vitro, did not negatively impact the healthspan of worms, but may increase worm median survival (+23.08%, p = 0.0286). The results of ASW compounds on worm survival in liquid media are summarized in Table 4.4.1.1. 155

Table 4.4.1.1 ASW Compound effect on C. elegans median survival and maximum lifespan in liquid media (Preliminary survival assessment)

Summary pilot lifespan experiment with ASW compounds. Median lifespan was determined by GraphPad Prism Software; maximum lifespan was calculated by the mean lifespan of the oldest 10% cohort of each group. P-values were determined from the Kaplan-Meier survival analysis for the entire population for

c the complete lifespan. Abbreviations: n, number of worms used; SEM, standard error of mean; , compared to control.

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Based on this pilot experiment, we decided to select the candidate compounds ASW03, ASW07 and ASW22 for further survival validation, as they demonstrated promise in extending worm survival. In addition to these compounds, ASW08 and ASW21, which consistently prevented loss of G6PD activity in vitro were included in these lifespan experiments. The G6PD inhibitor ASW25 was also included in the subsequent lifespan analyses to validate the effect on worm survival. The next set of survival assays were conducted on solid NGM agar, as opposed to the initial liquid-based survival analysis used in the pilot experiment, due to the reduced number of experimental groups enabling a more laborious technique for lifespan assessment, and the variability of the results obtained from the initial experiments in liquid culture. The control group from the pilot experiment had a longer than expected lifespan, which may be due to a combination of culture in liquid medium or from the use of heat-treated OP50 food stock. Furthermore, maintenance of nematodes on solid medium allows for efficient and better sensitivity in worm survival scoring, handling and lowered risk of contamination. The drugs for subsequent lifespans were refreshed on days 5, 10 and 15 of worm adulthood, with worms being transferred individually onto freshly made drug treatment plates in the presence of FuDR to prevent egg laying.

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Figure 4.4.1.2 The G6PD-stabilising small molecule ASW03 improves worm survival (A) ASW03 treatment at 50 µM improves worm survival. Little to no changes were observed in worms treated with 10 µM ASW03 compared to control group, with moderate improvement in worm healthspan observed in the presence of the drug at 50 µM. Data represents at least four independent experiments, each of which demonstrated a consistent effect (individual data in appendix). (B) 50 µM ASW03 increases worm median survival in N2 worms. Worms treated with 50 µM ASW03 had improved median survival (21.5 ± 0.646 days, + 15.59%, p = 0.0031) compared to vehicle-control worms (18.6 ± 0.509 days), with mild but not significant improvement observed following treatment of 10 µM. (C) ASW03 increases worm maximum lifespan at both 10 µM and 50 µM concentrations. Worm lifespan is extended in the presence of ASW03 at 10 µM (27.04 ± 0.583 days, +4.81%, p = 0.0312) and 50 µM (28.74 ± 0.785 days, +11.40%, p = 0.0395) compared to controls with a maximum lifespan of 25.8 ± 0.821 days. Maximum lifespan of each worm cohort was determined by averaging the maximum lifespan of the oldest 10% in the cohort. The graphs shows the median survival ± s.d. from five (10 µM concentration) or four (50 µM concentration) independent experiments. A minimum of 100 worms were used per condition for each experiment with at least four independent experiments performed.

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Compound ASW03, a robust G6PD stabiliser in vitro, increased the survival of drug-treated worms only at the 50 µM dose, with no changes in worm health observed at the lower 10 µM concentration (Figure 4.4.1.2.A). This improved worm healthspan was observed across at least four independent experiments, with approximately 100 worms per treatment group, and all treatments having been assessed in a blinded manner. Treatment with ASW03 at 50 µM raised the median survival of worms by 15.59% (p = 0.0031, n = 425) compared to control worms, which had a median survival of 18.6 ± 0.509 days (determined by five independent cohorts, n = 530). These worms also exhibited increased average maximum lifespan by 11.40% (p = 0.0395) during treatment at 50 µM (Figure 4.4.1.2.B and C), compared to the control maximum of 25.8 ± 0.821 days (determined by five independent cohorts, n= 530). Despite the lack of change observed in worm median survival following drug treatment at the lower dose of 10 µM, at 50 µM ASW03 marginally improved the maximum lifespan of the by 4.81% (p = 0.0312, n = 482) across five independent experiments (Figure 4.4.1.2.B and C).

The small molecule ASW08, which also stabilised recombinant G6PD in vitro was tested for its ability to alter C. elegans health and lifespan. At a decreased dose of 10 µM, the drug had a mildly detrimental effect on C. elegans survival, with more worm death occurring in early observations compared to the control group (Figure 4.4.1.3.A). However, this detrimental effect appeared to be negated with drug-treated worms having mildly improved survival compared to the control post-20-day worm adulthood with no significant changes observed in average maximum lifespan (Figure 4.4.1.3.C). The median survival of worms treated with 10 µM ASW08 had a non-significant trend towards improved lifespan, with treated worms having a median survival of 20.3 ± 0.668 days (p = 0.1124) compared to vehicle control of 18.6 ± 0.509 days (Figure 4.4.1.3.B). Increasing the dose of ASW08 to 50 µM greatly improved the survival of treated worms by 18.28% (22.0 ± 0.577 days, p = 0.0055) compared to control, and also resulted in a trend of improved worm maximum lifespan (Figure 4.4.1.3.B and C), although this observation was not statistically significant.

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Figure 4.4.1.3 50 µM G6PD-stabilising small molecule ASW08 improves worm median survival. (A) ASW08 may be mildly detrimental to worm health in early life at 10 µM concentration, but beneficial later in life and at a higher dose of 50 µM. Reduced worm survival was observed initially when treated with 10 µM of drug ASW08, stabilising and resulting in mild improvement of survival after 20 days of observation, whilst 50 µM ASW08 had a positive outcome on worm lifespan. Data shown is a summary of three independent experiments, individual experiments in Appendix. (B) 50 µM ASW08 increases worm median survival by 18.28% (22.0 ± 0.577, p = 0.0055, n = 299) compared to control (18.6 ± 0.509, n = 530), with mild but not significant improvement at the 10 µM concentration (20.3 ± 0.668, p = 0.1124, n = 333). (C) Treatment ASW08 shows trend in improving worm maximum survival in a dose-dependent manner. ASW08 when tested at 10 µM and 50 µM concentrations exhibited trend in increasing maximum lifespan of worms with increasing drug dosage, without statistical significance. Maximum lifespan of each worm cohort was determined by averaging the maximum lifespan of the oldest 10% in the cohort. The graphs shows the median survival ± s.d. from three independent experiments. A minimum of 100 worms were used per condition for each experiment.

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Figure 4.4.1.4 Small molecule G6PD stabiliser ASW22 improves worm lifespan and healthspan. (A) ASW22 improves worm healthspan and lifespan in a dose-dependent manner, with the drug at dose 50 µM having better healthspan increase than at 10 µM. The survival curve was collated from results from at least four independent experiments. (B) 50 µM ASW22 significantly increased worm median survival by 14.52% (21.3 ± 0.629, p = 0.0223, n = 451) compared to the control group. 10 µM ASW22 showed trend of improving the median survival of the worm, but this was not significant. The graphs shows the median survival ± s.d. from at least four independent experiments. (C) Both concentrations of ASW22 increased maximum lifespan of the worms, with a dose-dependent increase observed. 50 µM ASW22 increased maximum lifespan by 17.48% (30.31 ± 0.762, p = 0.0103) compared to control which has a maximum lifespan of 25.8 ±0.821 days, whilst 10 µM ASW22 extended maximum lifespan by 8.29% (27.94 ± 0.664, p = 0.0037). Maximum lifespan of each worm cohort was determined by averaging the maximum lifespan of the oldest 10% in the cohort. A minimum of 100 worms were used per condition for each experiment.

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The G6PD stabiliser ASW22 improved worm health and lifespan in a dose- dependent manner, with a modest improvement observed at 10 µM. ASW22 had a greater improvement present with an increased dose of 50 µM (Figure 4.4.1.4.A). This dose- dependent improvement was observed in both median and maximum survival, with 10 µM ASW22 showing mild but not significant increases in median worm median survival by 10.75% (20.6 ± 0.678 days, p = 0.0632, n = 576) whilst 50 µM ASW22 significantly increased median survival by 14.52% (21.3 ± 0.629 days, p = 0.0223, n = 451) compared to the DMSO vehicle control group which has a median survival of 18.6 ± 0.509 days (n = 530) (Figure 4.4.1.4.B). The maximum lifespan of ASW22 treated worms were also robustly improved, with an increase in maximum lifespan observed in the presence of both tested concentrations of 10 µM and 50 µM with a dose-dependent increase evident. Worms treated with 10 µM ASW22 had increased lifespan of 27.94 ± 0.664 days (p = 0.0037, n = 576), 8.29% increased compared to the control group. The increased dose of 50 µM ASW22 showed a greater lifespan extending effect on the worms, with a 17.48% (30.31 ± 0.762, p = 0.0103, n = 451) increase in total maximum lifespan observed in treated worms compared to control observed (Figure 4.4.1.4.C).

Compound ASW25, identified to be a potent G6PD inhibitor was also subjected to further testing in vivo in C. elegans to observe the effects of G6PD inhibition on the health and survival of drug treatment in animals. As with previous compounds, the effects of ASW25 on survival were assessed at concentrations 10 µM and 50 µM, with no difference in both median and maximum lifespan observed with both tested drug concentrations observed (Figure 4.4.1.5.A, B and C). A separate G6PD stabiliser also identified from the G6PD activator screen, ASW21, was also subjected to a single experiment for its ability to alter worm survival. ASW21 treatment significantly improved median survival when treated at 50 µM, resulting in a 23.66% increase in worm median survival compared to control in a single study, but was detrimental to worm maximum lifespan at 10 µM drug dose (Supplementary 13). The results from all lifespan studies conducted are tabulated and summarized in Table 4.4.1.2.

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Figure 4.4.1.5 Small molecule G6PD inhibitor ASW25 has no effect on worm survival and healthspan. (A) ASW25 at concentrations 10 µM and 50 µM do not significantly affect worm survival. The survival curve presented is collation of results from two independent experiments. (B) G6PD inhibitor ASW25 does not alter worm median survival when compared to the vehicle control group. The graphs shows the median survival ± s.d. from two independent experiments. (C) Both concentrations of ASW25 at 10 µM and 50 µM did not affect worm maximum lifespan. Maximum lifespan of each worm cohort was determined by averaging the maximum lifespan of the oldest 10% in the cohort. A minimum of 100 worms were used per condition for each experiment.

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Table 4.4.1.2 ASW Compound effect on C. elegans median survival and maximum lifespan

Summary pilot lifespan experiment with ASW compounds. Median lifespan is determined by GraphPad Prism Software; maximum lifespan was calculated by the mean lifespan of the oldest 10% cohort of each group. P-values were determined from the Kaplan-Meier survival analysis for the entire population c for the complete lifespan. Abbreviations: n, number of worms used; SEM, standard error of mean; ,

compared to control.

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Compounds ASW06 and ASW07, which shares remarkably similar molecular structures that differ at only a single atomic position were also subjected to further testing for their ability to extending or improve lifespan in C. elegans, with a greater focus in comparing the changes resulting from treatment in worms between the two drugs. Both compounds were identified from the screen as stabilisers of the G6PD enzyme, with modest enzyme activity preservation occurring in the presence of both drugs. In preliminary survival studies, drug ASW06 was detrimental for the median survival of worms, with a reduction of 15.38% observed in the median survival of worm compared to the control group (Table 4.4.1.1). In contrast, ASW07, which differs to ASW06 by only a single atomic change, was beneficial to median survival, with a 23.08% increase in median survival observed (Table 4.4.1.1). Both molecules were detrimental towards maximum lifespan. To gain a better understanding of how this small change between the two otherwise identical molecules alters and affects the outcome and characteristics of the drugs, the two compounds were also subjected to further animal studies via the assessment of C. elegans survival, alongside other lead compounds as described in Table 4.4.1.2. Both compounds were trialled at concentrations of 10 µM, as described in the section Experimental methods 4.3.3 Caenorhabditis elegans survival assay.

Both small molecules ASW06 and ASW07 stabilise and preserve the in vitro activity of recombinant human G6PD wild-type enzyme. However, when administered to C. elegans at concentrations of 10 µM, only ASW07 treatment resulted in better survival and lifespan. Worms treated with compound ASW06 showed no significant difference in survival compared to the control group, whilst ASW07 treated worms had a modest but significantly improved survival (p = 0.0020) (Figure 4.4.1.6.A). No changes in worm median survival were observed in ASW06 treated worms, with the median survival of both control and ASW06 treated worms remaining at 18.6 ± 1.14 days and 18.0 days respectively, with no significant difference between the two groups. Worms treated with ASW07 had a trend in increased median survival of 21.0 days but this was not significant (Figure 4.4.1.6.B). There was no significant difference in maximum lifespan between the control groups and any of the drug treatments. However, there is a trend of decreased maximum lifespan observed following treatment with ASW06, and a possible increase in maximum lifespan with ASW07 treated worms (Figure 4.4.1.6.C).

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Figure 4.4.1.6 A single atomic change between G6PD stabilisers ASW06 and ASW07 affect compound efficacy in modulating worm lifespan. (A) Worms treated with 10 µM ASW06 showed no significant difference in lifespan compared to the vehicle control group, whilst worms cultured in the presence of 10 µM ASW07 significantly increased the survival of treated worms (p = 0.0020). This was determined from two independent lifespan analyses of worms, with more than 80 worms assessed per group. Statistical analyses was performed using log-rank (Mantel-cox) test. (B) Compound ASW06 did not have an effect on median survival of treated worms, whereas ASW07 showed a trend in improved median survival of worms. (C) No significant changes were observed between control group and 10 µM ASW06 or ASW07 treated worm groups, but ASW06 shows trend in being detrimental for worm maximum lifespan whilst ASW07 potentially being beneficial for worm maximum survival. Maximum lifespans of each worm cohort were determined by averaging the maximum lifespan of the oldest 10% in the cohort. A minimum of 80 worms were used per condition for each experiment.

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The G6PD stabilisers ASW03, ASW08 and ASW22 were beneficial to C. elegans lifespan and were assessed for antioxidative capacity against the control antioxidants ascorbic acid and N acetyl cysteine to determine if the small molecules functions as direct antioxidants resulting in the observed lifespan extension of the animals. Using the CUPRAC antioxidant capacity assessment (Apak, Guclu, Ozyurek & Karademir, 2004), all three assessed small molecules ASW03, ASW08 and ASW22 exhibited little to no reductive capacity unlike the positive controls trialled within the experiment ascorbic acid and N acetyl cysteine which had a antioxidative capacity rate of 0.710 Trolox equivalent antioxidant capacity (TEAC) and 0.230 TEAC respectively (Figure 4.4.1.7).

G 6 P D A c tiv a to r s sa m p le r e d u c in g c a p a c ity in T r o lo x e q u iv a le n t (µ M )

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Figure 4.4.1.7 Antioxidant capacity assessment of lifespan extending G6PD stabilisers. Small molecules were assessed for antioxidant capacity with antioxidants ascorbic acid and N acetyl cysteine used as controls, and antioxidant capacity represented as trolox equivalent in µM. The antioxidant ascorbic acid had antioxidative capacity of 0.710 Trolox equivalent antioxidant capacity (TEAC), whilst N acetyl cysteine, another known antioxidant had an antioxidative capacity of 0.230 TEAC. The small molecules ASW03, ASW08 and ASW22 exhibited no antioxidative capacity even at concentrations of up to 1 mM..

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4.4.2 Improved lifespan observed in ASW compound-treated nematodes is not paired with an improvement in physical activity or healthspan

Most animals experience a decline in muscle strength, coordination and motility with normal ageing, creating an important need to improve not only the total lifespan of an organism, but also the healthspan during ageing to achieve longevity. As in with humans, C. elegans ageing is accompanied by a deterioration in muscle function, with studies showing the rate of age-related decline in activity being a good predictor of lifespan. A significant change and predictor of ageing in C. elegans is the active pharyngeal pumping rate (Chow, Glenn, Johnston, Goldberg & Wolkow, 2006; Glenn et al., 2004; Huang, Xiong & Kornfeld, 2004). Therefore, changes in pharyngeal pumping rates of the nematodes were compared to determine whether the improved lifespan of drug-treated animals were paired with improved health.

The pharyngeal pumping rates of worms treated with the DMSO vehicle were compared to those treated with the ASW compounds that had previously shown to increase the median survival of worms, namely, ASW03, ASW08 and ASW22. Compounds were again tested at concentrations of both 10 µM and 50 µM. As with the survival assay experiments, animals were exposed to drugs from Day 1 of worm adulthood to the end of the experiment, and all groups were maintained in the presence of 100 µM FuDR to prevent worms from laying eggs and with non-heat treated OP50 as a food source. As before, compounds were refreshed by transferring the worms onto freshly made agar plates containing the drugs on Day 5, 10 and 15 of worm adulthood.

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Figure 4.4.2.1 Age-associated decline in pharyngeal pumping ability of C. elegans is not improved or rescued by lifespan-improving ASW compounds. (A) Pharyngeal pumping rate of C. elegans treated with ASW03, ASW08 and ASW22 at concentrations 10 µM and 50 µM, with drugs refreshed on Day 1, 5, 10 and 15 of worm adulthood (n>10/group/timepoint), DMSO concentration maintained at 0.1%. (B) Comparison of pharyngeal pumping rate between DMSO control worms and worms treated with ASW03, (C) ASW08 and (D) ASW22. Statistical significance were determined by multiple t-test.

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An age-associated decline in pharyngeal pumping rate was observed in all treatment groups, with a steady decline in the number of pharyngeal contractions being observed from the age of day 5, and progressing until the day 17, when the experiment was halted due to worm death and lack of visible pharyngeal pumping from aged animals in all groups (Figure 4.4.2.1.A). As these compounds extended lifespan in previous experiments, both in terms of median survival, and in the case of compound ASW22, maximal lifespan, we hypothesised that an improvement in healthspan of the nematodes treated with the compound would also benefit from the treatment. This was not the case for the compounds ASW03, ASW08 and ASW22 at both tested concentrations, with no reduction in the declining pharyngeal pumping rate being observed with all treatments (Figure 4.4.2.1.A). A mild decline in pharyngeal pumping rate was observed with worms treated with 10 µM ASW03 when the worms were at day 8 of age, but no other significant differences between treatment groups and control groups were otherwise observed (Figure 4.4.2.1.B).

Mild differences in the contraction rates were observed in the early ages of nematodes treated with compound ASW08 at both 10 µM and 50 µM concentrations (Figure 4.4.2.1.C), but these changes were no longer present throughout the remaining lifespan, with no significant differences between treatment groups and control group observed at the later stages of worm life. Nematodes treated with the compound ASW22 exhibited a slight decrease in pharyngeal pumping rates at Day 8 of worm treatment (Figure 4.4.2.1.D), but not at any other stages of life. As these changes were only observed at isolated stages of life, the compounds were concluded to not affect the physical activity of animals. The improvement in worm survival following treatment with G6PD stabilisers was likely not due to a change in food intake or muscle contraction of the worm, which remains unchanged in worms that had been treated with the drug.

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Figure 4.4.2.2 ASW compounds improve lifespan of C. elegans but do not prevent or rescue age- induced loss of physical activity measured by thrashing rate. (A) Number of thrashes, with a complete sigmoidal movement of the worm in S complete medium scored as a single thrashing motion, was used to measure the physical activity of C. elegans in the presence of compounds at 10 µM and 50 µM, refreshed on Day 1, 5, 10, and 15 of worm adulthood and compared to DMSO vehicle-control. (B) Comparison of thrashing rate between DMSO vehicle treatment and worms treated with ASW03, (C) ASW08, and (D) ASW22. There were slight decreases in activity observed when worms were treated with ASW22 at 10 µM concentrations at the early stages of worm adulthood, with activity increasing mildly on day 10 adulthood, but no significant changes in activity were observed at the later stages of worm life. Statistical analyses were conducted using multiple t-test [* p<0.05, **<0.01, ***<0.001].

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Assessing physical movement through observing the number of worm thrashes, defined by the number of complete sigmoidal motions made by the worm while suspended in liquid, was used as a measure of worm motility and to assess changes in healthspan. The number of thrashes made by the suspended worms declined with age, with a steady decrease in worm thrashing observed from day 1 of adulthood until day 20 when the experiment was concluded. Mild variations were observed when worms were cultured in the presence of G6PD-stabilising ASW compounds, with no significant changes occurring between all groups (Figure 4.4.2.2.A). The presence of the G6PD stabilisers ASW03, ASW08 and ASW22 did not reduce the gradual decline of worm thrashing number as the worms aged, with no difference observed between all groups as well as when compared to the control group (Figure 4.4.2.2.A). Comparisons of thrashing rates between the control group and worms treated with 50 µM ASW03 showed mild variations, with drug-treated worms having mildly reduced activity at Day 1 adulthood (Figure 4.4.2.2.B). There was however no significant difference in worm activity between vehicle and drug-treated worms at both 10 µM and 50 µM concentrations for the remaining lifespan.

Animals treated with ASW08 at 50 µM showed slight improvements in activity during middle adulthood, with significant activity increases measured on day 5 and 10 of worm adulthood. The increase in activity ceased during old age, with no difference observed between the control and 50 µM drug-treated group from day 15 onwards (Figure 4.4.2.2.C). A slight decrease in activity was also measured on the first day of worm adulthood in worms treated with the decreased dose of 10 µM ASW08, with no changes in activity observed throughout the remainder of the experiment. Worms treated with ASW22 showed a trend of mild reduction of activity at both the 10 µM and 50 µM concentrations, and a significant reduction observed at the 10 µM concentration on day 1 and 5 of worm adulthood (Figure 4.4.2.2.D). A small, sharp increase in activity was also observed in worms treated with 10 µM of the drug at day 10 of worm adulthood, but the differences in worm activity was absent during the later stages of worm age (Figure 4.4.2.2.D).

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4.4.3 ASW compounds extends lifespan in a G6PD-dependent manner, but do not rescue age-associated decline of G6PD activity

Both G6PD enzyme activity and PPP activity are well known to decline with age in humans (Maurya, Kumar & Chandra, 2016; Niedermuller, 1986; Rodgers, Lichtman & Sheff, 1983). The basis of this project has been that this decline in the PPP causes disease and dysfunction from impaired NADPH generation, causing deficiencies in redox maintenance, reductive fatty acid biosynthesis, and infection defence. Impaired PPP activity might also cause deficiencies in ribose-5-phosphate levels, necessary for nucleotide synthesis and cell growth. This age-dependent decline in G6PD activity had been shown previously to occur in fruit flies (Hall, 1969), but with little other evidence of this decline available for other animal species. Here, we measured C. elegans whole body G6PD enzyme activity at various stages of worm life and determined that there was an age-dependent decline in G6PD activity.

C . e le g a n s w h o le b o d y G 6 P D e n z y m e a c tiv ity d e c lin e s w ith a g e

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Figure 4.4.3.1 Age-dependent decline of C. elegans G6PD enzyme activity. G6PD enzyme activity declined with age, with significant reduction in activity occurring on day 8 of worm adulthood by 17.88% compared to aged Day 1 worms, and further decline occurring as the worms age with age 11 day worms having 50.05% less activity than the control and worms more than 14 days of age having less than 60% enzyme activity than young worms. Statistical significance were determined by one-way ANOVA, with more than 150 worms utilized per group for G6PD enzyme activity measurement [** p<0.005, **** p<0.0001] (# two biological replicates, three technical replicates; ## one biological replicate, three technical replicates).

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This age-dependent decline in G6PD activity observed in C. elegans occurred from day 8 of worm adulthood by 17.88% compared to young Day 1 aged worms, which were used to represent 100% G6PD activity. A further decrease in enzyme activity occurred as the worm aged, with activity dropping by 50.05% activity by age 11 day, and worms 14 and 17 day of age having 39.94% and 39.04% total G6PD enzyme activity compared to young worms respectively (Figure 4.4.3.1). G6PD activity measured in C. elegans were exclusive of the eggs typically generated and housed within worms, which has also been determined to not contribute greatly to the overall G6PD activity levels of the worm (Figure 4.4.3.2). This was achieved by culturing the worms in the presence of 100 µM FuDR to prevent egg formation and egg laying.

G 6 P D a c tiv ity : w o r m e g g s v s . w h o le w o r m

0 .0 0 5

0 .0 0 4

m r

o 0 .0 0 3

u o

h 0 .0 0 2

U A 0 .0 0 1

0 .0 0 0 W ho le w o rm E xtra c te d e g g s

Figure 4.4.3.2 Eggs within gravid adult C. elegans contributed minimally to the overall G6PD activity of whole worms. Eggs within Day 1 gravid adult worms were isolated utilising the worm bleaching solution protocol and were subjected to the same worm lysate preparation prior to G6PD activity measurement. The G6PD activity present in the extracted eggs were compared to G6PD activity of lysate isolated from homogenized Day 1 gravid adult worms containing eggs. G6PD activity was determined by measuring the rate of total NADPH production within the sample and subtracting NADPH production by 6PGD to yield G6PD activity. There is minimal G6PD activity produced by the eggs present in gravid worms compared to G6PD measured in a whole worm.

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The compounds ASW03, ASW08, and ASW22 were beneficial for the survival of C. elegans worms, with extensions in median and maximum lifespan when these compounds were administered to animals at the post-development stage (Result section 4.4.1 Lifespan of C. elegans is improved with ASW compound treatment). As there is an age-dependent decline in G6PD activity worms, with activity dropping to half of that of a young worm during old age (Figure 4.4.3.1), and these compounds are known stabilisers of recombinant G6PD enzyme in vitro, we hypothesised that the improvement of lifespan observed occurs via preservation of G6PD enzyme activity in old age. The addition of the G6PD stabilisers are hypothesised to stabilise and preserve G6PD activity, resulting in preservation and elevation of G6PD levels during old age, which will result in increased G6PD activity. C. elegans worms were grown in the presence of compounds ASW03, ASW08 and ASW22 at 50 µM, the concentration which consistently exhibited robust lifespan and survival improvement in treated worms across more than three independent studies, with G6PD activity assessed at day 1 (young adults), day 5 (mid- age) and day 13 (old age) of lifespan. All animals were grown in the presence of 100 µM FuDR to minimize egg laying for ease of handling, and worms were individually picked and collected for G6PD measurement.

Treatments with the compounds ASW03, ASW08 or ASW22 post-development in C. elegans did not rescue the age-related loss of G6PD activity, with no significant difference in G6PD activity observed between treatments at all ages tested (Figure 4.4.3.3.A). There was a trend towards increased G6PD activity observed in the young day 1-old worms in the drug-treated worms, but this increase was not significant. No significant changes were observed between control and drug-treated worms at any stages of the worm life, and between all drug treatments (Figure 4.4.3.3.B, C and D), with treatment of ASW03, ASW08 or ASW22 not restoring or preserving any age-dependent G6PD enzyme activity loss.

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Figure 4.4.3.3 Lifespan-extending ASW compounds do not improve or rescue age-associated loss of G6PD activity. (A) G6PD activity of worms treated with vehicle-control DMSO or ASW compounds at young-adult (Day 1), mid-adult (Day 5) or old-adult (Day 13) age. Whole worm G6PD activity was determined by subtracting 6PGD activity from whole worm NADPH production by both G6PD and 6PGD enzyme. Addition of compounds ASW03, ASW08 or ASW22 did not rescue the age-related loss of G6PD activity in worms. (B, C, D) Comparison of G6PD activity between vehicle-control treated worms and worms treated with compounds ASW03, ASW08 or ASW22. There were no significant changes observed between control and drug-treated worms at any stages of the worm life. Treatment with any of the ASW compounds did not restore aged-associated reduction of G6PD.

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As G6PD activity was not rescued by the administration of G6PD stabilisers during old age, we decided to assess whether this improvement in lifespan does occur in a G6PD-dependent manner, despite activity of the enzyme not being preserved or elevated during old age. Stress resistance of worms in the presence of these compounds were assessed using the oxidants diamide and paraquat. Diamide, an oxidant probe for thiols, depletes the NADPH reservoir in a glutathione-dependent manner (Kosower & Kosower, 1995). Cells with reduced amount of NADPH are unable to regenerate thiol which may result in stress or lethality (Kosower, Zipser & Faltin, 1982). Paraquat (1,1’ -dimethyl- 4,4’ -bipyridylium dichloride) undergoes an NADPH-dependent reduction to form free radicals which react with molecular oxygen to produce a superoxide anion, eventually resulting in the production of H2O2 which have the potential to cause lipid peroxidation and eventual stress-induced death (Smith, 1985).

Compounds ASW03, ASW08 and ASW22 are predicted to increase G6PD activity by promoting stabilisation of the enzyme in an active dimer/tetrameric form, resulting in increased NADPH formation, which may assist in regeneration of reduced glutathione, thus conferring resistance against diamide-induced oxidative stress. In contrast, animals treated with the ASW compounds are predicted to be more susceptible to paraquat-induced stress due to increased G6PD-dependent NADPH formation, as paraquat relies on the presence of NADPH to generate its toxic form. By determining the survival of drug-treated worms in response to these two oxidants, we can predict the effect of these G6PD stabilisers on NADPH. In summary, we would anticipate that increased NADPH generation from G6PD would result in resistance to G6PD, and increased susceptibility to paraquat. The concentrations of oxidant diamide and paraquat were determined via the conduct of a dose-response experiment with varying dosage of diamide and paraquat, with worms being monitored up to 6 days post- oxidant exposure.

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Figure 4.4.3.4 Oxidative stress resulting in worm lethality was induced at 200 mM diamide and 100 mM paraquat. Worms were subjected to oxidant treatment for 2.5 hours to induce oxidative stress by diamide or paraquat with increasing dose to determine the minimum concentration of oxidant required to induce toxicity in the worm. (A) Diamide concentration 150 mM and lower were not sufficient in inducing lethal stress in C. elegans, whilst concentrations above 200 mM resulted in near complete death. At 200 mM diamide concentration, 60.19% worms were scored alive on day 6 of monitoring. (B) Paraquat concentrations more than 100 mM resulted in complete death of oxidant treated worms, with 48.57% worms remaining on day 6 of monitoring.

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Through a dose-response study of varying concentrations of diamide and paraquat, a 2.5 hour insult of diamide at 200 mM was determined to be sufficient in inducing oxidative damage to worms without resulting in irreversible worm death, allowing for possible rescue via pharmacological interventions such as G6PD stabilisers (Figure 4.4.3.4.A). Treatment with diamide at 150 mM or lower was not sufficient to induce lethal stress in C. elegans worms, with only 11.69% worm death occurring at 6 days after oxidant exposure. Exposure to diamide at concentrations above 200 mM resulted in more than 97.67% worm lethality at the 6-day mark, and 200 mM diamide resulted in 39.81% death (Figure 4.4.3.4.A). As worms exposed to concentrations of diamide above 200 mM do not recover from an oxidative insult, treatment at 150 mM and lower did not induce stress-induced lethality. As a result, exposure to 200 mM diamide for 2.5 hours was determined to be ideal to assess the effect of ASW compounds in the diamide-induced stress response of C. elegans worms.

Paraquat exposure for 2.5 hour at a concentration of 100 mM was sufficient to induce worm lethality, without being so harsh that the exposed worms do not recover from the insult, with 65.71% worms remaining alive 2 days after oxidant exposure (Figure 4.4.3.4.B). Concentrations of paraquat above the 100 mM dose resulted in near complete worm lethality by day 6 of monitoring, with 51.43% worm death occurring by day 6 following 100 mM paraquat exposure (Figure 4.4.3.4.B). Paraquat exposure at 200 mM was fatal to worms, with >99% worm death occurring by day 2 after the oxidant exposure. Thus, the dose of diamide at 200 mM and paraquat at a mildly increased concentration of 125 mM were determined to be ideal oxidant concentrations to induce sufficient oxidative damage to the worms, whilst allowing for resistance or increased susceptibility towards oxidative stress to be determined following pharmacological interventions of the ASW compounds. Concentrations of 125 mM and 200 mM were used for both oxidants, with the exposure time of 2.5 hour being kept constant to study the effects of ASW compound treatment on the worm stress resistance. Worm survival were scored at least once every 2 days, with emphasis on monitoring on day 3 and day 5 after oxidant exposure.

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Figure 4.4.3.5 ASW compound treated C. elegans exhibited increased resistance to low-dose diamide- induced stress and increased susceptibility to high dose, but not mild paraquat-induced lethality. C. elegans treated with ASW compounds were assessed for changes in stress resistance against oxidants diamide and paraquat at both high and low oxidant doses or 200 mM and 125 mM respectively. (A) C. elegans were treated with 125 mM paraquat, which requires NADPH and GSH to be metabolised, therefore increased mortality expected with increased G6PD activity. (B) Treatment with diamide, a thiol oxidant that depletes GSH pools, with decreased mortality expected with increased G6PD activity. (C) Exposure to a high dose paraquat insult of 200 mM for 2.5 hours.

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Table 4.4.3.1 C. elegans stress resistance assay against diamide and paraquat result summary

Summary stress resistance assay with ASW compounds against oxidants diamide and paraquat. P-values were determined from the ANOVA one-way analysis for the entire population at the given day.

c Abbreviations: n, number of worms used; SEM, standard error of mean; , compared to control.

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C. elegans treated with the G6PD-stabilising compounds ASW03, ASW08 and ASW22 displayed improved survival in both median survival and maximum lifespan (Figure 4.4.1.2, 4.4.1.3, 4.4.1.4 respectively). In worms treated under the same conditions, an improvement in resistance against oxidative stress induced by paraquat at a high dose of 200 mM was observed. An oxidative insult of low dose paraquat of 125 mM resulted in significant worm death in the control group, with 74.70% worms surviving the insult when monitored on day 3 after the oxidant exposure, and more death following with only 55.42% worms surviving on day 5 post-stress induction (Figure 4.4.3.5.A). A similar trend was observed in compound-treated worms, with ASW03 and ASW22 treated worms having no significant difference when compared to the survival of the untreated control group. ASW08 treated worms displayed mild but significant protection 3 days after the insult, with 90.60% of the population surviving the oxidant insult, but no difference compared to the control group on day 5 post-exposure (Figure 4.4.3.5.A).

When the paraquat concentration was raised to 200 mM, there was increased mortality with 31.68% of the control group being scored alive 3 days after the insult, and 22.77% remaining on day 5. ASW03, ASW08 and ASW22-treated animals were more susceptible to the increased paraquat dose, with significant decreases in worm survival being observed on day 3 and day 5 after the insult (Figure 4.4.3.5.C). These results are summarized in Table 4.4.3.1. Treatment with ASW compounds treatment improved the survival of C. elegans exposed to diamide-induced oxidative insult, with the vehicle- control group survival being decreased to 76.74% and 55.81% 3 days and 5 days post- exposure respectively, whilst ASW03, ASW08 or ASW22 compound-treated groups having >95% survival and >87.01 survival on day 3 and day 5 respectively (Figure 4.4.3.5.B).

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4.5 Discussion

Lead compounds identified from the high-throughput small molecule screen described in Chapter 1 appeared to increase G6PD enzyme activity when co-incubated with substrates. As described in Chapter 2 however, we discovered that these molecules exhibited characteristics more in line with being enzyme stabilisers, preserving enzymatic activity and preventing activity loss rather than increasing enzyme activity per se. A series of in vitro assays on recombinant wild-type G6PD enzyme showed that the compounds ASW03, ASW07, ASW08, ASW13 and ASW22 reliably prevented a loss in G6PD activity with time, with the treatment groups often displaying maintaining baseline activity compared to the vehicle control, which declined in activity with time spent at room temperature. As G6PD activity declines with age (Rodgers, Lichtman & Sheff, 1983), we hypothesised that these G6PD stabilising compounds could enable G6PD activity to be maintained into old age, thus preventing a decline in G6PD activity leading to an improvement in overall healthspan in animals. The aim of these experiments was to have pharmacological activators of G6PD which would mimic the effects of G6PD overexpression, which are beneficial for lifespan and healthspan (Legan et al., 2008; Nobrega-Pereira et al., 2016).

Promising G6PD-stabilising candidates were tested for their ability to maintain lifespan and healthspan using the well-studied model organism Caenorhabditis elegans, which were chosen for their short lifespan, low cost, low maintenance, ease of handling and ability to generate genetically homogenous populations. This was necessary for our purposes due to the considerable number and unknown bioavailability of the novel G6PD- stabilising small molecules, thus requiring the drugs to be trialled at varying concentrations. Furthermore, recombinant human wild-type G6PD enzyme were used within the G6PD HTS described in Chapter 1 to screen for small molecules that altered enzyme activity. The NADP+ binding region of the G6PD enzyme is highly conserved across evolution, with the protein sequence for the dinucleotide-binding region GASGDLA believed to be important for NADP+ binding within the enzyme being completely reserved between humans and C. elegans (Bautista, Mason & Luzzatto, 1995; Kletzien, Harris & Foellmi, 1994). For this reason, it is expected that small molecules which increase G6PD enzyme activity in the human variant of the enzyme have a high possibility of having the same effect on the C. elegans G6PD homologue. 183

G6PD stabiliser trial to identify leads for lifespan extension

C. elegans were grown in the presence of the treatment after development, i.e. from adulthood, and were monitored for survival as well as physiological changes. The DNA synthesis inhibitor 5-fluorouracil-2’-deoxy-ribose (FuDR) was introduced to the C. elegans cohort just prior to the worms reaching sexual maturity, which allowed for a synchronous population to be maintained without any known changes to post- maturational development, behaviour and appearance (Mitchell, Stiles, Santelli & Sanadi, 1979). The use of this drug allowed for ease in worm handling and reduced egg formation and hatching during cohort maintenance, allowing for larger number of groups to be handled in a single experiment and reduces the risk of contamination by offspring worms. This reduction in worm offspring also prevents the rapid depletion of the OP50 food source, thus requiring fewer plate changes within experiment for the restoration of the cohort food source.

An initial worm survival analysis was performed with treatments being introduced to the animals in one single dose upon worm adulthood, revealing several lead candidates to focus on for further testing. This was necessary as each survival experiment requires a significant length of time for completion and are highly time consuming. With 22 different G6PD activators having been identified through the G6PD HTS as described within Chapter 1, each with a unique molecular structure and possible mode of action on the enzyme, the focus was limited to several promising leads due to time and workload constraints. Furthermore, the lifespan analyses were conducted in the presence of positive controls for extended lifespan in C. elegans such as doxycycline hydrochloride (Supplementary 12), which is well known to be a pharmacological intervention that extends worm lifespan (Ye, Linton, Schork, Buck & Petrascheck, 2014) and the known G6PD inhibitor dehydroepiandrosterone (DHEA), which shortens worm maximum lifespan (Supplementary 12). These controls were put in place to validate the results of the survival analyses and to ensure pharmacological interventions are able to cause an observable effect on the worm lifespan.

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The pilot worm survival assay assessing the effects of pharmacological intervention using small molecules was performed with 8 select ASW compounds out of the pool of small molecules isolated from the G6PD HTS. These included 7 compounds that best stabilised the G6PD enzyme when tested in vitro, and 1 G6PD-inhibiting compound that reduced recombinant protein activity. These compounds were tested at concentrations of 10 µM and 50 µM, with both concentrations shown to alter enzyme activity when tested in vitro on recombinant enzyme. Compounds were not tested at higher doses to avoid issues of solubility, limited availability of the drugs, and due to lifespan-extending pharmacological interventions having previously been shown to function effectively in the lower micromolar range (Ye, Linton, Schork, Buck & Petrascheck, 2014). All treatments were maintained with DMSO solvent concentration kept constant at 0.2%, with worms maintained in liquid media instead of solid media as described previously (Solis & Petrascheck, 2011). The choice to maintain the worms in liquid media was made as it allowed for larger number of treatments to be tested in a single experiment, reduces the loss of animals with each transfer cycle and allows for the heat-treated OP50 food source to be replenished when required.

The positive control, whereby worms were treated with a single dose of the antibiotic doxycycline exhibited improved median survival and maximum lifespan in the pilot experiment, with a large increase in median survival of up to 84.62%. This validated the survival assay, as doxycycline is a known pharmacological intervention which extends C. elegans lifespan (Ye, Linton, Schork, Buck & Petrascheck, 2014). Worms treated with the G6PD inhibitor DHEA had decreased maximum lifespan, falling in line with our hypothesis, whereby an improvement in G6PD enzyme activity will prove beneficial to the worm lifespan, whilst a decrease in G6PD activity results in shortened survival. However, molecule ASW25 which inhibits the enzyme activity of recombinant G6PD in vitro did not negatively affect the survival of C. elegans, instead mildly improving worm median survival in contrast to our hypothesis, which may be due to off- target drug interactions benefiting worm lifespan, or inadequate drug dosage resulting in a possible hormesis effect (Cypser & Johnson, 2002). Control animals which were exposed to 0.2% DMSO exhibited a median survival of 13 days and had a maximum lifespan of approximately 38 days, which was noticeably longer than when animals are maintained on solid agar, as has been reported previously (Solis & Petrascheck, 2011).

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Based on our hypothesis, improvement of G6PD activity following treatment with G6PD-stabilising small molecules should result in better healthspan and survival of C. elegans. A single treatment with the compound ASW03 at the start of worm adulthood, previously shown to consistently prevent time-dependent loss of activity of recombinant enzyme in vitro, improved median survival in worms without differences observed between the two trialled drug doses. Another G6PD-stabiliser of interest was ASW22, which at 50 µM showed a trend towards improved worm median survival, although this result was not statistically significant, with no significant changes observed in worm maximum lifespan following treatment with either G6PD-stabilisers. Other G6PD- stabilisers identified from the G6PD HTS were ASW13, ASW15 and ASW21, and unlike small molecules ASW03 and ASW22 which mildly increased lifespan, , were detrimental to the survival of C. elegans with a negative impact on both median and maximum survival following treatment. This was not unexpected due to vast differences in molecular structures of these compounds despite all trialled molecules exhibiting G6PD- stabilising characteristics and good predicted binding affinities with the structural NADP+-binding pocket of the G6PD enzyme, with these differences in structure possibly altering drug-enzyme amino acid interaction within this enzyme region. There may also be differences in drug bioavailability or potency, resulting in different survival outcomes of the treated animals.

The G6PD-stabilisers ASW06 and ASW07 differ from one another by only a single atomic position, resulting in a change of a furan to thiophene on the otherwise identical molecular structure. Both were detrimental to the worm total lifespan in the preliminary lifespan study, with treatments shortening maximal lifespan of C. elegans. However, an improvement in median survival was observed in worms treated with these drugs at a lower concentration of 10 µM, with a non-significant trend observed following ASW06 treatment, and a significant improvement with ASW07 at the 20 µM concentration (Table 4.4.1.1). The worms in these groups had improved survival earlier in life, but decreased survival in later life. With these results, compounds ASW06 and ASW07 were worthy of further exploration. Compound ASW07 was never trialled at 50 µM due to limited availability of the compound, as well as the reduced concentration of the drug stock. As the concentration of DMSO in all groups were kept constant at 0.2% and the ASW07 stock was at 10 mM concentration, ASW07 could not be trialled at a higher concentration. 186

The G6PD-stabilisers ASW03, ASW06, ASW07 and ASW22, which were determined by the preliminary lifespan study to be promising lifespan-extending drugs, were chosen and subjected to experimental repeats for the validation of the results. Compounds ASW13, ASW15 and ASW21 were dismissed from subsequent experiments due to time constraints and the limited number of groups to be pursued. Compound ASW08, which is another G6PD-stabiliser that was not included in the initial lifespan analyses was also added into subsequent survival assays as the small molecule proved to be a consistent and efficient stabiliser of the enzyme in vitro as determined by experiments conducted at later stages. Subsequent survival analyses were conducted with the maintenance of worms on solid NGM agar, as opposed to the liquid-based assay used in the pilot experiment. Maintaining worms on solid media is more labour intensive and requires more stringent handling of the animals during lifespan assessments, thus reducing the number of groups able to be handled at any given time. However, this method allows for better efficiency and sensitivity in worm survival scoring and a lowered risk of contamination, which were problems that arose during the use of the liquid-based worm handling.

Narrowing our focus to the few drugs of interest resulting from the pilot experiment, we were able to assess the effect of the G6PD stabilisers on worm health and lifespan more efficiently and with much greater sensitivity, reducing error that may have arisen with the handling of a large number of treatment groups. For example, the control group in the pilot experiment had a longer than expected lifespan which may have been due to a combination of culturing the worms in the liquid medium, which may have resulted in increased worm lifespan, contamination or from the use of heat-treated OP50 food stock which may affect bacterial proliferation and reduce bacterial infection resulting in lifespan extension (Garigan, Hsu, Fraser, Kamath, Ahringer & Kenyon, 2002). With worm maintenance on solid medium, live OP50 may be provided to the worms as a food source without risk of contamination or bacterial growth saturation which hinders visibility of the worm in culture and depletion of food source is easily observed and may be replenished as needed. In subsequent survival analyses, the drugs were made fresh into the NGM agar of which the worms were maintained on, with the drugs being refreshed on day 5, 10 and day 15 of worm adulthood by manually transferring the worms onto freshly made agar on the specified days. This was to ensure the worms were continuously exposed to the drugs of which the half-life and stability had 187 not yet been established. As with the first experiment, animals were also maintained in the presence of 100 µM FuDR to prevent egg laying, with all treatments assessed in a blind manner to eliminate bias during worm handling.

ASW small molecule G6PD stabilisers are beneficial to survival

The small molecule ASW03 faithfully stabilised recombinant G6PD enzyme when trialled in vitro and prevented a loss of enzyme activity over time. C. elegans maintained in the presence of the compound were longer lived compared to untreated controls, with improved worm healthspan observed across four independent experiments. The worms were exposed to the drug at two concentrations, 10 µM and 50 µM, with the increase in survival only being observed with the higher dosage, suggesting either poor intake or absorption of the drug by these animals, or reduced drug potency. The working concentration of 50 µM however is appropriate for C. elegans survival studies (Ye, Linton, Schork, Buck & Petrascheck, 2014). Despite the lack of change observed in the worm median survival following treatment at the lower dose of 10 µM, there was a slight but significant increase in worm maximum lifespan which were validated across five independent experiments. The EC50 of ASW03 determined from the log dose screen performed as described in Chapter 2 was 6.55 ± 1.76 µM. This suggests that at this concentration, the drug elicits half the maximal activity increase of the enzyme in vitro but may be considered mild when administered to C. elegans via diffusion from the maintenance agar due to C. elegans having a thick collagenous outer cuticle of which may hinder the penetration of the drugs into the worm (Lints & Hall, 2009).

To gain a better understanding as to how compound ASW03 improves the survival of the treated worms, functional assays were conducted on aged worms maintained in the presence of the drug. As most animals experience a decline in muscle strength, activity and motility with age, we decided to assess if the lifespan extension resulting from treatment with 50 µM ASW03 had coincided with improvements in the healthspan of worms during old age. These were assessed via the measurement of worm thrashing as a determinant of worm motility, and through measuring rates of pharyngeal pumping. Treatment with 50 µM ASW03 improved survival as observed by the increase of median survival and maximum lifespan of treated worms, but do not result in any changes to the

188 motility and movements of the worms. Thus, the cause for the lifespan extension observed following treatment with ASW03 remains unknown, with additional experiments required to explore the benefits that follow with the treatment. G6PD activity loss during old age was also not rescued, with an age-related decline of G6PD activity after day 5 of worm adulthood occurring despite the presence of 50 µM of the drug. As drug ASW03 is a faithful stabiliser of G6PD enzyme as determined by in vitro studies, this lack of G6PD enzyme activity loss prevention is surprising, and suggests the improved survival following ASW03 treatment may be the result of off-target effects.

The G6PD stabiliser ASW08 also improved both median survival and maximum lifespan, with a dose-dependent trend being observed with drug treatment, and worm median survival following treatment of 50 µM being statistically significant following three independent trials. Compound ASW08 was determined to have an EC50 of 7.24 ± 0.23 µM when trialled on recombinant G6PD enzyme as described in Chapter 2 and may have a better outcome on lifespan when compared to drug ASW03 despite being similar in drug efficacy. As described earlier, the trialled drugs concentrations in C. elegans are likely to result in much lower concentrations being exposed to the animal due to the exogenous collagenous cuticle of the worms. Given this, the result of the low dose drug treatment on worm lifespan through agar diffusion is remarkable. The observed improvements of median survival at the higher ASW08 dose was not accompanied by prevention of activity loss that comes with old age as measured by the thrashing rate and pharyngeal pumping, with activity having been monitored on the first, fifth and thirteenth day of worm adulthood. An age-dependent decline in G6PD activity was also not rescued in the presence of the drug, despite the drug stabilising the enzyme in in vitro trials. Therefore, like compound ASW03, the cause of the survival improvement observed with the drug effect remains unknown and requires further investigation.

Drug ASW22 robustly improved survival when introduced to C. elegans worms more efficaciously compared to other drug candidates, with ASW22-treated worms having a positive outcome in survival in both median survival and maximum lifespan. This survival benefit was dose-dependent, with statistically significant improvements to maximum lifespan observed at both concentrations, and in median survival following high dose drug treatment. The method by which ASW22 improved worm survival remains unknown, with no significant changes to worm motility and activity observed as

189 measured by worm thrashing and pharyngeal pumping throughout the worm lifespan, similar to the other trialled G6PD-stabilisers ASW03 and ASW08. These worms were observed to be healthier, appearing youthful with increased activity compared to the control counterparts through visual observation during general worm handling, which was evident despite the treatment groups being blinded. Worm activity and measures of ageing will have to be better quantified through the use of other functional parameters such as the measurement of egg laying as a measure of reproductive capacity to better understand how the drug causes this lifespan improvement in worms. Treatment with the higher dose of the drug did not change the G6PD activity of the worms throughout lifespan, with no changes in G6PD activity observed when compared to day 1, day 5 and day 13 control counterparts. This lack of change in G6PD activity, despite drugs ASW03, ASW08 and ASW22 being G6PD enzyme stabilisers could be due to off-target effects by the drugs, or the drugs could be affecting the enzyme in other ways to increase enzyme activity. This may also be caused by experimental procedures, whereby the worms were washed at least three times with buffer to remove bacteria and in doing so may have resulted in the removal of the drug as well. These drugs may be interacting with enzymes in a transient manner, and their removal during sample processing could result in the absence of the drug during G6PD activity measurements.

ASW06 and ASW07 share near identical structures but have different effect on C. elegans survival

Compounds ASW06 and ASW07, which differ in structure by only a single atomic change at an attached five carbon ring, were by chance separately identified from the G6PD HTS. Both drugs modestly preserved recombinant G6PD activity in vitro with the drugs exhibiting similar EC50 and activity preservation on purified enzyme despite this small difference in structure. We hypothesised the drugs to have identical outcomes on lifespan during in vivo treatment due to their similarities, raising overall G6PD activity in worms during old age and thus resulting in lifespan extension. Both drugs were detrimental to C. elegans maximum lifespan when introduced to worms in the single dose pilot experiment, but mildly improved survival in early life with little difference noted between the two compounds and thus were subjected to additional investigation to better elucidate the survival improvement observed. Despite the structural and characteristic 190 similarities between drugs ASW06 and ASW07, these drugs exhibited slightly different effects on worm survival. No changes in worm median survival were observed following treatment with either drugs, with compound ASW07 exhibiting trend but non-significant improvement in worm maximum lifespan. However, ASW06 may be detrimental to worm survival, with a mild but non-significant decrease in worm maximum lifespan observed. Due to the lack of availability of these compounds, this experiment was only repeated twice, and thus will have to be repeated for experimental replicate.

The effect of the drugs on worm health and activity were not explored for compounds ASW06 and ASW07 due to the lack of drug availability and will also have to be pursued in the future. Compounds ASW06 and ASW07 were also treated at a concentration of 10 µM, as the drug stock solutions were made at a concentration of 10 mM due to low quantities of the drug available for purchase and the need to maintain the DMSO solvent concentration at 0.1%. Therefore, trials conducted at higher dose will have to be performed to observe for any changes that occur to worm lifespan, as treatment at 10 µM may be too mild to elicit a response. Other G6PD stabilisers such as ASW03, ASW08 and ASW22 were more effective when treated at a higher dose in worms, with the higher concentration of the drug eliciting significant lifespan improvement in worms. Treatment with lower concentrations of test compounds showed a non-significant trend towards increased lifespan. Both ASW06 and ASW07 may be beneficial to lifespan when treated at higher concentrations such as that seen with the other G6PD stabilisers.

G6PD inhibitor ASW25 does not affect worm survival

Although the main focus of the project was to identify small molecules which increases G6PD enzyme activity, several small molecules which inhibited G6PD enzyme were also isolated to be used as a control for the study and to also be pursued as potential therapies for G6PD inhibition. Only one drug was chosen to be further explored for the effects of G6PD inhibition in C. elegans worms, which is compound ASW25. This compound exhibited the strongest and most consistent G6PD inhibition when tested in vitro on recombinant enzyme, which was then administered to worms at doses of 10 µM and 50 µM. As increased G6PD activity is known to improve lifespan in organisms such as mice (Nobrega-Pereira et al., 2016) and fruit flies (Legan et al., 2008), we had expected

191 to observe a decrease in worm survival following G6PD inhibition like that seen with the G6PD inhibitor DHEA. Treatment with ASW25 at both concentrations did not significantly affect worm lifespan, with no changes seen to either median survival or maximum lifespan across two independent experiments. This lack of change could be due to drug instability during agar preparation, or could be an issue with drug dosage. This experiment will have to be repeated for validation, and ideally with a wider range of drug concentrations.

Small molecules improve C. elegans G6PD-dependent stress resistance

Worms treated at the young adult stage with the G6PD stabilisers ASW03, ASW08 and ASW22 had exhibited increased in median survival, maximum lifespan or both, particularly when compounds were administered at the higher dose of 50 µM. Treatment with these compounds did not alter physical activity and there was no apparent change in health, as measured by pharyngeal pump rates and motility, and the G6PD activity which declines during old age was not restored despite the drugs stabilising the enzyme and preventing activity loss when tested in vitro. Measurement of G6PD enzyme activity involved harvesting the worms and assessing the lysate for enzyme activity in vitro after sample processing which removes all drugs from the sample for a period of time before measurement. The absence of compounds during sample processing may provide a false picture of G6PD enzyme activity, especially if the drugs interacts with the enzyme transiently. G6PD enzyme activity was therefore assessed in vivo by comparing worm resistance to oxidative stress, as G6PD enzyme is the main source of NADPH which is crucial for oxidative stress defence.

The worms were exposed to acute stress induced by two different oxidants, diamide and paraquat, both of which relied on NADPH for their mode of action. Diamide depletes the NADPH reservoir in a glutathione-dependent manner, resulting in oxidative stress and eventual death of the organism. Paraquat undergoes an NADPH-dependent reduction to produce free-radicals eventually resulting in H2O2 which is harmful to the organism. Depending on the availability of NADPH, of which the G6PD enzyme is the main contributor, these oxidants will be either more, or less potent in inducing oxidative stress in the animal. As the ASW compounds are predicted to function by stabilising the

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G6PD enzyme, resulting in increased enzyme activity within the animal, treatment with the compounds should present an increased NADPH availability in the organism, which will confer resistance to diamide-induced oxidative stress and will increase the animal’s susceptibility to paraquat-induced oxidative stress.

C. elegans were treated with the small molecules for 24 hours before being acutely exposed to either oxidant and returned to drug-treated plates for recovery. The potency of the oxidants were measured by the number of surviving worms over the next few days. Drug-treated worms were exposed to paraquat of two different concentrations, a mild dose of 125 mM and the increased dose of 200 mM to compare for differences in survival. Diamide was also exposed to the worms at these two concentrations, however groups exposed to 200 mM diamide showed no loss in worm survival despite the high oxidant dosage possibly due to experimental/handling error or loss of oxidant potency. This increased diamide dosage was omitted from the study as low diamide exposure of 125 mM was sufficient in inducing oxidative damage to the animals, resulting in significant worm lethality. ASW03-treated animals exposed to low dose acute paraquat stress had mildly improved, but not statistically significant survival when scored three days after the oxidant exposure, with any differences in survival having been lost by day 5 of the experiment. Exposure to the higher dose of paraquat however resulted in increased worm death compared to the control, noticeable from a day after the oxidant exposure, with the treated worms also being resistant to diamide induced stress. Similar results were seen with ASW08 and ASW22 treatment, with the ASW08 drug treated worms having significantly improved survival initially after the low dose paraquat exposure, which by the end of the experiment showed no difference compared to the control group. Worms treated with ASW22 had no significant difference compared to the control group following exposure to low dose paraquat. The worms treated by both drugs exhibited increased susceptibility to 200 mM paraquat-induced death, and resistance to stress induced by diamide suggesting improved G6PD activity. Together, these results suggest that NADPH availability in the small molecule-treated worms are elevated, which correlated to G6PD activity.

Thus, there is evidence that treatment with the G6PD-stabilising ASW compounds may result in the increase of G6PD activity which presents itself in the resistance of the worms to oxidative stress, despite the lack of increased G6PD activity in cell extracts

193 following ASW compound treatments. This effect is unlike the recently identified G6PD stabilser AG1, whereby a reduction of ROS levels and improvement of GSH following the treatment with the enzyme stabiliser was coupled with a measurable increase in G6PD activity (Hwang et al., 2018). However, the stress response and G6PD activity measured in this study were assessed in response to an oxidative stress insult, whereas in our study G6PD activity was measured in the absence of stress. The ASW small molecules could be trialled in a similar manner as described by Hwang et. al, that is, to assess improvements in the stress response and G6PD activity of the animals following introduction of a stressor.

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Chapter 5 Chapter 5 General discussion

General Discussion

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5.1 Study significance

Within this project, we aimed to identify novel small molecules which would increase the pentose phosphate shunt by targeting the rate-limiting enzyme G6PD, which is situated at the start of the pathway and which plays a critical role in the intricate network of redox maintenance in aerobic organisms and is involved in crucial processes for aged cells. These include nucleotide synthesis for faithful DNA replication, and a source of metabolites for immune function in the form of NADPH, required by NADPH oxidase in granulocytes. Candidate molecules were identified through a small molecule screen for compounds that increased G6PD activity. These compounds were to be then used as a mode of PPP activation to increase NADPH to reduce oxidative damage and R5P for DNA synthesis and repair, and potentially resulting in an increase in lifespan or overall healthspan through the increase in NAD+ availability. G6PD-activating molecules may also be useful in the treatment of G6PD deficiency, which is the most common human enzyme deficiency. Conversely, inhibitors of the G6PD enzyme have been suggested as treatments of diseases associated with elevated G6PD activity including cancer and malaria.

It was hypothesised here that the decline in the activity of the PPP may play a role in cellular dysfunction during ageing and that pharmacological activation of G6PD, the first enzyme in this pathway, may represent a new method for treating biological ageing. The activity of the PPP decreases with age in liver, brain, kidney and skeletal muscles. In the brain, the PPP declines steadily with age, with little to no PPP activity observed at an age of 32 months (Niedermuller, 1986). This pathway supplies the cell with biochemical intermediates which we hypothesise are key challenges for ageing cells. There were no known pharmacological agents which activated the G6PD enzyme at the start of this project, and the aim of this project was to identify novel small molecules with drug-like properties which could modulate G6PD enzyme activity, potentially resulting in the development of a new class of geroprotective agents which may maintain health into old age.

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5.2 Evaluation of experimental direction

A high-throughput assay was developed and optimized to compare changes to G6PD enzyme activity following treatment with small molecules candidates, allowing for activators and inhibitors of the enzyme to be isolated from a large library of drug-like small molecules. Lead compounds isolated from the screen were then subjected to further studies, determining the characteristics of the molecules and their effects on the G6PD enzyme. These molecules, initially thought to function as enzyme activators which by definition are allosteric since they must allow the substrate to bind to the enzyme for a reaction to occur, were found to prevent destabilisation of the enzyme, and prevent enzyme activity loss rather than increasing enzyme catalysis. Computational docking simulations indicated favourable interactions between the stabilising molecules to the NADP+ binding regions of the enzyme, with preference for the substrate binding site believed to be important for maintaining the enzyme in its active dimeric state. Several validated leads were selected for further exploration, revealing mild changes to enzyme kinetics when cultured in the presence of G6PD-stabilising small molecules.

Drugs with strong stabilising features and potency as determined from in vitro studies were administered to C. elegans nematodes and were found to be beneficial to worm survival, possibly due to changes in stress resistance, with no significant changes to physical activity or motility of the animals. The experimental approach, direction and limitations will be discussed in this section, with focus on the main aim of assay development for the identification of G6PD enzyme activators, characterization of the lead small molecule and the choice of the animal model used in this project.

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5.2.1 High-throughput small molecule screen

10,240 drug-like molecules from the WECC-CCIA diversity library were randomly generated and screened for agents which increased G6PD enzyme activity. A direct, quantitative absorbance-based G6PD enzyme activity assay as previously described (Tian et al., 1998) was chosen to compare the changes to G6PD enzyme activity following incubation with the small molecules and optimised for processing the drugs in a high-throughput scale. This measurement method was chosen due to its sensitivity and low cost of materials and reagents for the assay, only requiring the enzyme substrates G6P, NADP+ and the recombinant protein. The small molecules were screened against commercially available recombinant human wild-type G6PD protein instead of a deficiency variant due to the highly polymorphic nature of the G6PD gene, with over 140 mutations or combinations of mutations having been reported (Beutler, 1991; Beutler & Vulliamy, 2002) and the interest of our project to identify activators which will increase enzyme activity to that above normal enzyme levels.

Twenty-eight novel compounds with drug-like properties were identified at the conclusion of the small molecule screen, twenty-two of which resulted in increased G6PD product NADPH of up to 167.88% above that of the control, and six which inhibited the G6PD enzyme down to 18.84% of control enzyme activity. Lead compounds which were commercially available were purchased for additional trials and characterisation, which resulted in the isolation of eight activating agents and two inhibitors for selection as molecular leads. These small compounds were initially hypothesised to be allosteric activators of G6PD enzyme, however upon further trials were revealed to instead operate as enzyme stabilisers, preventing the loss of enzyme activity with time. The identification of small molecule enzyme activators is considered a challenge in the field of drug discovery and development (Zorn & Wells, 2010), and at the start of this project there were no known pharmacological agents for direct activation of G6PD enzyme. The identification of these eight G6PD stabilisers may therefore represent an important contribution to the field.

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5.2.2 ASW drug G6PD enzyme activity modulation

G6PD-stabilising small molecule candidates were subjected to further characterization using computational molecular docking simulations to better understand the mode of action on the modulation of wild-type G6PD enzyme activity. Drug enzyme binding affinity represented by Goldscore binding fitness were determined using the BIOVIA Discovery studio software version 4.1, revealing favourable binding affinity of the G6PD activity modulating ASW small molecules to the structural NADP+ binding pocket of the enzyme, with this particular region believed to be involved in the maintenance of long-term stability of the G6PD enzyme (Wang, Chan, Lam & Engel, 2008a), keeping the enzyme in its active dimeric/tetrameric form (Au, Gover, Lam & Adams, 2000). These results were supported by enzyme-stabilising features of the lead compounds as exhibited by in vitro studies on purified recombinant enzymes. These molecules also exhibited strong binding affinity for the second NADP+ binding region, with preference for the structural NADP+ binding site.

Despite the favourable binding observed by most small molecules to the structural NADP+ binding region of the enzyme, drugs ASW03 and ASW13, both of which displayed potent G6PD enzyme-stabilising features, exhibit poor binding efficiency to this region. Poor drug-enzyme Goldscore fitness suggesting weak binding affinity does not always indicate a lack of interaction of a drug to the assessed region of the enzyme, as the molecules could be interacting with proteins on a transient basis, eliciting a change in enzyme conformation resulting in a change in enzyme behaviour. Compound ASW03 was also subjected to preliminary drug-enzyme binding assessment by Surface Plasmon Resonance, which showed a binding affinity between the drug and wild-type G6PD protein in the tens of micromolar range. With that said, computational simulations only provide estimates and predictions of enzyme-drug interactions, with the results requiring confirmation by other experimental means. In future work, x-ray crystallography studies of the interaction between these small molecules and G6PD protein would provide further insights into the mechanism of action and options for chemical optimisation of these leads. This work is currently ongoing with collaborators.

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Enzyme activators are typically thought to induce an allosteric change since they must allow the substrate to bind to the enzyme for a reaction to occur. Interestingly, G6PD contains two binding sites for the NADP+ substrate, one as a regulatory or structural binding domain for enzyme dimerization, and the other as a catalytic binding site. Using in silico binding studies, these small molecules were predicted to preferentially interact with the structural substrate binding domain involved in enzyme dimerization. As a result, these candidate molecules may not function in a typical allosteric manner, and may instead be involved in promoting dimer formation. Several amino acids of interest were identified following analyses of the drug-enzyme computational interactions predictions, with all molecules identified from the G6PD enzyme activator screen having interactions with residues Arg357, Lys366, Arg370, Arg487, Phe501 and Thy503 in the NADP+ binding region. This suggests these residues play a role in the enzyme stabilisation. A single key amino acid which only interacted with the molecules which resulted in elevated enzyme activity and no interactions with any of the inhibiting molecules or the NADP+ substrate control was the lysine residue situated at amino acid Lys497. This suggests a possible role of this particular amino acid in the activation or increased stabilisation of the enzyme, and suggests a possible target for future pursuit for molecular interventions which improve G6PD enzyme stability.

Receptor-ligand docking simulations could be used to develop drug-like molecules to target Lys497, which could be involved in increased enzyme stabilisation, whilst having interactions with all key amino acids as identified from the screen to generate candidate molecules for the prevention of enzyme instability. Due to time constraints with this project and the supporting results obtained from the computational modelling for the enzyme-drug interaction, an in-depth assessment to define the action of the ASW G6PD-stabiliser was not performed. There are however ongoing projects being conducted by collaborators to determine the pharmacological and biochemical action of these small molecules. Experimental confirmation of the importance of this site could be performed by mutating K497 to a negatively charged residue, for example a K497E mutation, and testing whether this mutant protein is insensitive to stabilisation by these small molecules.

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Recombinant versions of the two most common G6PD deficiency variants, G6PD A-- and G6PD Mediterranean (Beutler, 1994) were also constructed, expressed and purified to determine if the lead G6PD-stabilising ASW molecules ASW03, ASW06, ASW07 and ASW22 were able to prevent activity loss by increasing enzyme stability of the G6PD mutants as they do for the wild-type enzyme. The G6PD-stabiliser ASW08, which improved nematode survival was not trialled on the deficient G6PD protein due to the drug being unavailable at the time of the experiment. ASW03, ASW06 and ASW07 mildly improved the activity of G6PD A-- following a brief period of enzyme activity loss, with ASW22 showing non-significant trend in reducing the loss of activity. The molecules ASW03 and ASW06 showed mild improvement in enzyme activity of both deficiency variants, with the activity preservation being more pronounced with the G6PD A-- protein. It is possible that G6PD Mediterranean, being a Class I deficiency variant having <10% enzyme activity compared to wild-type (WHO, 1989), the severity of the activity impairment could not be salvaged through the use of pharmacological intervention.

As most mutation variants differ from the wild-type G6PD by a single amino acid change, and there are more than 140 variants G6PD variants having been described (Beutler, 1994; Cappellini & Fiorelli, 2008), these proven G6PD-stabilising molecules could be trialled on other purified recombinant G6PD variants to narrow down key amino acids involved with the activation of the enzyme, as a majority of the G6PD deficiency variants differ from the wild-type enzyme by only a single amino acid. Similarly, deletion of amino acids on the G6PD enzyme could be performed using site-directed mutagenesis to isolate key amino acids which interact with these compounds.

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5.2.3 Choice of animal model

The nematode Caenorhabditis elegans was used as a model organism for ageing studies in this project. C. elegans are a widely used animal model in the study of pharmacological interventions on ageing due to their relatively short lifespan, rapid generation time and well-characterised genetics (Blagosklonny, Campisi & Sinclair, 2009; Braeckman & Vanfleteren, 2007; Fontana, Partridge & Longo, 2010). Furthermore, G6PD is highly conserved across evolution, with the G6PD enzyme having a completely conserved amino acid region corresponding to the substrate binding site (Au et al., 1999; Bautista, Mason & Luzzatto, 1995; Kletzien, Harris & Foellmi, 1994), where the G6PD- stabilising ASW compounds were predicted to target. There are however limitations to the use of this animal model, with limited insights on changes that occur to the healthspan of animals following exogenous treatments. Measurements of pharyngeal pumping rate and body bend locomotion rate are common behavioural assays used in the assessment of C. elegans health (Hart, 2006) and were used in this project as markers of healthspan. There were no significant changes to worm pharyngeal pump rate or motility, which may be due to lack of assay sensitivity, or lack of changes occurring to animal healthspan following treatment with the lifespan-extending ASW small molecules.

The use of additional animal models such as mice would allow for a better understanding of the beneficial outcomes of ASW small molecules, which faithfully extended lifespan in C. elegans. This would allow for validation of the lifespan extension in other species. Furthermore, mammalian G6PD sequences have been aligned using conventional algorithms and were shown to have ~94% sequence identity. When compared to lower vertebrates, invertebrates and microorganisms, this sequence identity decreases as expected, but never decreases below 20% (Notaro, Afolayan & Luzzatto, 2000). Thus, trialling small molecules on a mammalian model such as in mice would be much more translatable to humans. Mice that are deficient in G6PD have significantly higher levels of oxidative stress markers in the brain (Jeng, Loniewska & Wells, 2013), which is believed to predispose to neurodegenerative diseases (Behl & Moosmann, 2002; Halliwell, 2001; Markesbery & Carney, 1999). This presents a possible avenue for use of the G6PD-stabilising compounds in reducing free radical damage by increasing PPP activity. C. elegans used in the present project are a commonly used model for the study of pharmacological intervention on longevity and physical performance in vivo, but do

202 not provide strong functional assays for brain function. The G6PD-stabilising ASW compounds could be trialled in a more appropriate model organism such as mice to assess changes to brain function and cognition, and it would be of interest to trial these compounds in the treatment of oxidative stress-induced neurodegenerative disorders.

Prolonged treatment with these compounds in mice would also validate the clinical relevance of these lifespan extension results, and allow for the study of functional parameters such as endurance, assessment of changes to cognitive functions and changes to motor functions such as balance and strength. At present, several G6PD-stabilising ASW small molecules are being administered to Drosophila melanogaster fruit flies in an ongoing survival experiment, with results estimated to be available mid-2019. G6PD deficiency has also been suggested to play a role in fertility and pregnancy, with G6PD deficient women experiencing increased spontaneous abortions (Toncheva & Tzoneva, 1985) and reduced G6PD activity in mice resulting in smaller litter sizes (Nicol, Zielenski, Tsui & Wells, 2000; Tsai, Schulte, O'Neill, Chi, Frolova & Moley, 2013). This relationship was also observed in C. elegans worms (Yang et al., 2013) and it would be of interest to trial the ASW drugs to observe for improvements to reproduction and embryogenesis in worms following treatment with the G6PD-stabilising drugs. Assessment of worm reproduction following drug treatments were not conducted in this project due to time constraints but may be worth pursuing in the future.

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5.3 Clinical relevance of G6PD stabilisers

Three novel small molecules ASW03, ASW08 and ASW22 were identified from the project and found to faithfully prevent destabilisation of the G6PD enzyme, reducing activity loss with time in vitro. The molecules exhibited favourable binding to the NADP+ binding regions of the G6PD enzyme, with preference for the structural NADP+ binding domain believed to be involved in keeping the enzyme in its active dimeric/tetrameric state. When administered to C. elegans nematodes, these compounds improved survival, with mild lifespan extension which could be attributed to changes occurring to the G6PD enzyme activity resulting in a change to stress resistance of the nematodes. This aligns with our hypothesis, whereby increased G6PD activity may result in an improvement to lifespan and healthspan as observed in transgenic animals over-expressing this enzyme (Legan et al., 2008; Nobrega-Pereira et al., 2016).

There were no known small molecules to directly activate G6PD directly at the commencement of the project. However, recent work from the Rosen-Mochly lab has since identified a small molecule activator of the G6PD enzyme, AG1, which stabilises the G6PD enzyme in its active dimeric/tetrameric state as well as increasing the activity of wild-type, the Canton mutant as well as several other common deficiency variants of the enzyme (Hwang et al., 2018). From this study, the AG1 small molecule not only increased the activity of G6PD, but decreases oxidative stress in zebrafish, cells and stress-induced erythrocytes, with effects on animal lifespan and healthspan not being explored. This work is encouraging as we had also hypothesised our ASW compounds to function via stabilisation of the enzyme, but with no evidence of increasing enzyme catalysis observed. This study also highlights the need for the identification of drugs targeting G6PD for the management of G6PD enzyme deficiency, which is the most common human enzyme defect (Beutler & Vulliamy, 2002). Compared to this study, our project may have priority for the identification of small molecules influencing G6PD activation to improve animal lifespan and healthspan instead of just targeting the enzyme deficiency.

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In this project, pharmacological interventions resulting in increased G6PD activity were hypothesised to be beneficial to the lifespan and healthspan of animals. Consistent with this, the newly identified G6PD stabilisers ASW03, ASW08 and ASW22 extended lifespan when administered to nematodes. Despite there being a wealth of experimental evidence correlating G6PD activity and survival, with G6PD activity being known to decline during ageing (Biagiotti, Malatesta, Capellacci, Fattoretti, Gazzanelli & Ninfali, 2002; Duncan, Dell'orco & Guthrie, 1977; Magnani, Stocchi, Bossu, Dacha & Fornaini, 1979) and over-expression of the enzyme resulting in enhanced longevity (Legan et al., 2008; Nobrega-Pereira et al., 2016), it is unknown if this relationship is applicable to humans. With human longevity being highly polygenic and multifactorial, there are no correlation between G6PD deficiency and longevity in humans, with studies observing increased mortality with the genetic deficiency (Petrakis, Wiesenfeld, Sams, Collen, Cutler & Siegelaub, 1970), and others showing no association between the two (Heller, Best, Nelson & Becktel, 1979). Unfortunately, there is little data on the health of individuals carrying Class V G6PD variants, where enzyme activity is elevated. The last known variant reported was variant G6PD Hektoen in 1969, with a single amino acid substitution resulting in change in enzyme regulation and increased levels of the variant protein (Dern, McCurdy & Yoshida, 1969; Yoshida, 1970). Longevity assessments of Class V G6PD individuals would provide some insights into the relationship between G6PD enzyme activity and survival.

The pentose phosphate pathway is the sole NADPH-generating source in mature red blood cells, which lack a citric acid cycle (Takizawa, Huang, Ikuta & Yoshida, 1986) and have a long lifespan after losing their capacity for protein synthesis (Mason, 1996), making erythrocytes more susceptible to haemolysis in instances where G6PD enzyme is deficient. There are three major red blood cell disorders, sickle cell anaemia, α- thalassemia and G6PD deficiency, all of which are characterized by increased susceptibility to oxidative stress which accelerates the destruction of red blood cell resulting in lysis (Chan, Chow & Chiu, 1999). The G6PD-stabilising molecules identified here may be of use in treating these erythrocyte-related disorders, preventing lysis and thus alleviating symptoms of these diseases. It has been previously suggested that the activity of red blood cell enzymes may have a compensatory mechanism, whereby in populations where G6PD is deficient, such as with old age or in the case of thalassaemia, red blood cell production increases to compensate for this loss either caused by 205 intrinsically decreased G6PD activity or by the shortened lifespan of the erythrocytes (Bernini et al., 1964). The efficacy of the G6PD-stabilising molecules in treating these blood disorders are therefore debateable.

The G6PD-stabilising small molecules identified in this project may also be useful in the treatment of age-related diseases such as cardiovascular disease and cancer, as ROS is believed to be involved directly or indirectly with the development of cancer (Hercberg, Galan, Preziosi, Alfarez & Vazquez, 1998). NADPH generated from the PPP functions directly and indirectly as an antioxidant (Kirsch & De Groot, 2001), which may reduce the accumulation of ROS. This may also prove useful in disease circumstances whereby there is are excess generation or accumulation of ROS, such as in diabetes, cardiovascular disease, neurodegenerative disorders or renal failure (Behl & Moosmann, 2002; Halliwell, 2001; Markesbery & Carney, 1999; Tiedge, Lortz, Drinkgern & Lenzen, 1997; Zhang, Apse, Pang & Stanton, 2000).

The high-throughput screen performed here also resulted in the identification of eight G6PD inhibitors, termed compounds ASW23 – ASW28. These inhibitors presented

IC50 values of <10.31 µM, with the strongest inhibitor, ASW23 having an IC50 value of 2.79 ± 0.25 µM and reduces the G6PD enzyme activity to 18.84% of the normal activity. The second-best inhibitor, ASW24 inhibited enzyme activity to 20.02% compared to the control with an IC50 of 1.04 ± 0.18 µM, which was the most potent inhibitor identified from the screen. The G6PD enzyme inhibitors identified from this work were similar in potency and efficacy to those identified recently (Mele et al., 2018; Preuss et al., 2013), but compared to known G6PD inhibitor DHEA, which has an IC50 of ~330 µM (Preuss et al., 2013), these small molecules are approximately 100 to 300 -fold more potent at inhibiting the enzyme. That being said, the reported IC50 values of DHEA varies between studies due to differences in enzyme purity and activity (Hamilton et al., 2012). The G6PD-inhibiting ASW small molecules identified from this project may therefore be more effective in treating diseases whereby G6PD activity is abnormally elevated.

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5.4 Future directions

In this project, novel small molecules were screened against commercially available purified recombinant human wild-type G6PD enzyme, identifying compounds which activate or inhibit wild-type enzyme activity. When tested against purified recombinant deficiency variants, the small molecules which functioned effectively against the wild-type enzyme did not have the same efficacy on mutant variants. The drug screen may be worth repeating against G6PD enzymes resembling common G6PD deficiency protein, such as the G6PD A-- and Mediterranean variants. G6PD A-- mutations are largely found in African populations, affecting approximately 10% of black Americans, and G6PD Mediterranean mutants are widespread in the middle east (Beutler, 1994; Cappellini & Fiorelli, 2008; Vulliamy, Mason & Luzzatto, 1992). This would allow the development of small molecules which could be used in the treatment or management of G6PD deficiency.

Compounds which increased lifespan extension in C. elegans should be tested again in a log dose manner, whereby varying concentrations of the drugs are introduced to the worms to determine the concentration at which the best improvement in worm survival is observed. Compounds for testing would be ASW03, ASW07, ASW08 and ASW22, which at the concentrations trialled caused a reliable increase in survival. In our lifespan experiments, animals were maintained in the presence of only two drug concentrations, that is 10 µM and 50 µM, which may not have been ideal concentrations for optimum survival but still resulted in lifespan extension. It is possible that a lower, or higher drug dose could result in a larger observed improvement in health and lifespan. Furthermore, several other G6PD-stabilisers did not extend lifespan when treated in worms which may be due to issues in compound uptake, metabolism or dosing. For example, ASW13 faithfully improves recombinant enzyme activity and prevents activity loss with time in vitro but was detrimental to worm survival when trialled at 10 µM and 50 µM. This drug could be too potent and may have resulted in toxicity at the concentrations tested here, which could be alleviated if a lower dose was used instead. Therefore, treatments with ASW compounds could be further explored in C. elegans by observing the effects of varying the dosage of these small molecules.

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Another G6PD-stabilising compound, ASW21 had also been tested for its ability to extend lifespan in C. elegans worms only once, with no experimental repeat having been conducted due to time constraints. Thus, the results are only preliminary with more repeats of this experiment required. Like the other G6PD stabilisers ASW03, ASW08 and ASW22 tested on the animals, ASW21 increased the survival of the worms when treated at the higher concentration of 50 µM at the median survival, with improvements also seen at the level of maximum lifespan but without statistical significance. A lower dose of ASW21 of 10 µM mildly, but significantly reduced worm maximum lifespan. Therefore, ASW21 may be of interest to pursue as a possible lifespan extending compound in worms, with more experimental replicates required to confirm these results. There were also a number of compounds that had not been tested in animals. The G6PD HTS yielded 28 candidate compounds, with 22 being G6PD-stabilisers and 6 inhibitors of the enzyme. Only 9 of the 28 small molecules were ever tested for the effects on C. elegans lifespan and healthspan, with the other compounds being placed on hiatus due to time constraints or the limited availability of the small molecules.

Due to time constraints of the project, the physiological and metabolic activity changes following treatment with the G6PD-stabilising ASW molecules were not explored in detail. Worms treated with these G6PD-stabilsers exhibited improved survival with evidence suggesting improvements in G6PD-dependent stress responses, despite a lack of G6PD activity improvement. Fluorescence Lifetime Imaging (FLIM), which measures the endogenous fluorescence of metabolitesincluding NADH/NAD+,

NADPH ratio and FAD/FADH2 via simultaneous multiphoton imaging, or mass spectrophotometry analyses of drug-treated extracts could allow for better elucidation of changes occurring within C. elegans or mammalian cells following treatment with the G6PD-stabilising molecules. These techniques could especially be useful in determining differences occurring to the NADP+/NADPH ratio of C. elegans which are long-lived following treatment with ASW compounds which should result in G6PD-dependent NADPH production.

Furthermore, the lifespan-extending small molecules were never shown to directly increase G6PD activity in isolated C. elegans crude extract, despite faithfully preventing activity loss of recombinant G6PD enzyme. However, these same molecules were proven to be beneficial to C. elegans survival and lifespan when cultured in the nematode growth

208 medium. This may be due to the crude cell lysate not experiencing the same enzyme activity loss compared to a purified enzyme sample, which may be due to a buffering system from high protein concentration, other enzyme-stabilising factors within the lysate or inactivation of the molecules by other components within a crude cell lysate. G6PD activity loss could be induced via dilution of the extract, increasing incubation time length, temperature or proteases to observe small molecule prevention of enzyme activity loss. These molecules may also have been trialled at sub-optimal dosage, as the drug characteristics, uptake and bioavailability remain unknown. To better understand the properties of these novel molecules, pharmacokinetic studies in a chosen organism, ideally mice for future functional studies and progression of this project, could prove very informative.

More in-depth computational studies and the use of medicinal chemistry to improve structure-activity relationships could also be used to improve these G6PD- stabilising ASW molecules pharmacologically and biochemically, yielding molecules with increased potency . The ASW small molecules should also be trialled for activating or stabilising characteristics against other NADP+-dependent enzymes, such as 6- phosphogluconate dehydrogenase (Rippa, Giovannini, Barrett, Dallocchio & Hanau, 1998), glyceraldehyde-3-phosphate (Kitatani et al., 2006), aldehyde dehydrogenases (Lindahl, 1992) to better understand and confirm the binding interaction of the small molecules with the NADP+-binding site. Furthermore, this interaction could be further explored via the use of nuclear magnetic resonance spectroscopy to monitor and observe intermolecular interactions between the ASW drugs and the G6PD enzyme (Pellecchia et al., 2008), or isothermal titration calorimetry (Ward & Holdgate, 2001) to elucidate the degree of enzyme stabilisation.

Erythrocytes are also known to undergo storage lesion, which is an existing concern in transfusion practice whereby structural and functional changes occurs as the red blood cells age within the storage solution (Garcia-Roa et al., 2017). This presents a potential use for these G6PD stabilisers to increase G6PD activity and reduce ROS levels to prevent haemolysis of stored erythrocytes, as evidenced by the use of the G6PD- activating small molecule AG1 (Hwang et al., 2018). ASW compounds could be trialled in a similar manner to determine if these molecules may be suitable candidates to prevent haemolysis of RBCs during storage and transfer.

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5.5 Conclusion

Here, we have performed a small molecule screen and identified at least four structurally-unrelated novel small molecules with drug-like properties which not only faithfully stabilise G6PD enzyme to prevent a loss in enzyme activity, but also improve the survival and lifespan of C. elegans worms following drug treatment, believed to occur in a G6PD-dependent manner. These molecules are predicted by molecular docking studies to interact with the enzyme at the structural NADP+ binding region, resulting in increased enzyme stabilisation and preservation of enzyme activity, with mild changes occurring to the enzyme kinetics. These new molecules demonstrate that G6PD can be modulated by pharmacological approaches and offer the opportunity for a new class of drugs that can delay biological ageing and possibly extend lifespan.

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Supplementary figures

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G 6 P D H T S o p tim iz a tio n : N A D P H sta n d a r d c u r v e

3 + C o n v e rte d N A D P

N A D P H

)

m n

U 2

3 (

0 0 2 0 4 0 6 0 8 0 1 0 0 A m o u n t (n m o l)

Supplementary 1 Standard curve to determine the absorbance value of converted NADP+ and NADPH at 340 nm. Substrate NADP+ which had been converted by G6PD enzyme under standard G6PD enzyme assay conditions to NADPH and pure NADPH were assayed at varying concentrations (0 – 80 nmol) to determine the absorbance value of the products when measured for absorbance at 340 nm. The substrate NADP+ was converted to NADPH by the addition of G6PD enzyme and substrate G6P to the reaction and allowing the conversion to proceed until saturation. There was a slight decrease in absorbance produced by the converted NADP+ compared to NADPH despite equal amounts of substrates being measured, with NADP+ conversion by G6PD enzyme to NADPH plateauing with amounts above 40 nmol NADP+. Substrate NADP+ amounts of 0 – 40 nmol produces a linear increase in absorbance after being converted to NADPH.

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Supplementary 2 Recombinant human wild-type G6PD enzyme activity in the presence of enzyme substrates G6P or NADP+. (A) NADPH production of G6PD enzyme cultured in the presence of substrates G6P or NADP+. The presence of either substrates G6P or NADP+ reduces enzyme activity loss up to time 30 minutes, with little to no enzyme activity detected from either group after 40 minutes of incubation. (B) G6PD enzyme activity of enzymes cultured in the presence or absence of substrate represented by area under the curve (AUC). The presence of substrates G6P or NADP+ during enzyme incubation significantly reduced enzyme activity loss, preserving enzyme activity with NADP+ substrate having a stronger preservation effect than G6P. Statistical analysis was performed using One-way ANOVA.

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A S W C o m p o u n d s e ffe c t o n r e c o m b in a n t h u m a n w ild -ty p e G 6 P D e n z y m e

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A S W 1 5

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Supplementary 3 Several small molecules identified from the G6PD HTS do not alter enzyme activity. ASW small molecules identified from the G6PD HTS were purchased from chemical distributors and were trialled for enzyme activity-altering properties by comparing enzyme activity at time 0 and 30 minutes, during which the G6PD enzyme experiences a gradual decrease in enzyme activity. The G6PD activity of the DMSO control at time 0 was used to represent enzyme activity at 100%. The small molecules ASW01, ASW02, ASW12, ASW15, ASW19, ASW20, ASW23, ASW26, ASW27 and ASW28 did not significantly alter the G6PD enzyme activity at both tested timepoints of time 0 or time 30 minutes, suggesting the lack of either enzyme-stabilising or inhibiting capabilities. The results were summarized and graphed from at least three independent experiments. Statistical analyses were conducted using One-way ANOVA.

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Supplementary 4 G6PD activators derived from the HTS screen (ASW01 – ASW06). EC50 values representing drug concentration to achieve half maximal response are presented as mean value with standard deviation.

215

Supplementary 5 G6PD activators derived from the HTS screen (ASW07 – ASW12). EC50 values representing drug concentration to achieve half maximal response are presented as mean value with standard deviation.

216

Supplementary 6 G6PD activators derived from the HTS screen (ASW13 – ASW18). EC50 values representing drug concentration to achieve half maximal response are presented as mean value with standard deviation.

217

Supplementary 7 G6PD activators derived from the HTS screen (ASW19 – ASW22). EC50 values representing drug concentration to achieve half maximal response are presented as mean value with standard deviation.

218

Supplementary 8 G6PD inhibitors derived from the HTS screen (ASW23 – ASW28). IC50 values representing drug concentration to achieve half maximal response are presented as mean value with standard deviation.

219

Supplementary 9 Expression of recombinant G6PD enzyme in BL21 (DE3) E. coli in the presence or absence of chaperone proteins. (A) SDS-PAGE of bacterial soluble fractions from recombinant G6PD expression with/without co-expression of vectors pRIL and pBB550 encoding chaperone proteins visualised via Coomassie staining. (B) Western blot for G6PD protein of SDS-PAGE containing bacterial soluble fractions of recombinant G6PD expression with/without co-expression of vectors. Fractions collected overnight after induction of protein expression yielded more G6PD protein, with co-expression of the protein with both chaperones pRIL and pBB550 greatly improving protein yield. Abbreviations: Empty, control with no G6PD protein expression; WT, recombinant G6PD_WT expression; pRIL, co- expression with pRIL vector encoding tRNA for arginine, isoleucine and leucine; pBB550, co-expression with pBB550 vector encoding chaperones DnaK, DnaJ, GroELS.

220

Supplementary 10 Mascot peptide summary report. Mascot compares the observed spectraÂ of a provided sample to a protein database which identifies the most likely matches, producing a peptide summary report. The most significant protein hits are as listed, with the chaperone GroEL/GroES indicated as the potential contaminant in the purified G6PD protein fraction.

221

Supplementary 11 G6PD/Small molecule interactions determined by Surface Plasmon Resonance (SPR). Steady state affinity between the tested small molecule and recombinant human wild-type G6PD enzyme were determined by plotting the response at each titration point once the equilibrium is reached, with the curve fitted and fixed using the values obtained from the NADP+ positive control obtaining KD values. Compound ASW03 had binding affinity for the G6PD enzyme of KD 18 ± 6 µM, with ASW08 exhibiting a KD of 40 ± 15 µM and ASW22 having KD of 12 ± 7 µM.

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Supplementary 12 ASW compound treatment on C. elegans survival in liquid media survival assay. ASW compounds were refreshed on Day 5, 10 and 15 of C. elegans worm adulthood and heat-treated OP50 food source refreshed as needed. (A) G6PD inhibitor DHEA significantly reduced worm median survival at concentration 50 µM, and decreased worm maximum lifespan at both tested drug concentrations. (B) Treatment with Doxycycline improved worm median survival and maximum lifespan significantly at 50 µM drug concentrations, with mild but significant improvement in median survival observed following 10 µM treatment. (C) Treatment with small molecule ASW15, which upon further testing did not stabilise G6PD enzyme in vitro did not affect worm health or lifespan. (D) G6PD-stabiliser ASW21 treatment at both 10 µM and 50 µM concentrations decreased worm median survival and maximum lifespan.

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Supplementary 13 The G6PD-stabilising small molecule ASW21 improves worm median survival at 50 µM drug dose but may be detrimental to worm maximum lifespan at 10 µM. (A) ASW21 at 50 µM concentration improves worm survival, with little to no changes observed to overall lifespan. (B) There were no significant changes to worm median survival at 10 µM ASW21 (19 days, n = 133), with significant increase in median survival observed following treatment with 50 µM ASW21 (+ 4.4 days compared to control, 23 days, n = 138). (C) ASW21 at 10 µM significantly decreased worm maximum lifespan from 27.15 ± 2.115 days to 24.62 ± 1.865 days (- 2.53 days, p = 0.0022), whilst 50 µM drug treatment resulted in no significant changes to worm maximum lifespan (27.36 ± 1.55 days). Maximum lifespan of each worm cohort was determined by averaging the maximum lifespan of the oldest 10% in the cohort. The graphs shows the median survival ± s.d. a single survival experiment. A minimum of 100 worms were used per condition for each experiment. Statistical analyses were performed using one-way ANOVA.

224

Supplementary 14 C. elegans survival assay trialling lead G6PD-stabilising ASW small molecules. Small molecules ASW03, ASW07 and ASW22 at 10 µM dose improved worm median survival significantly, with the positive control Doxycycline hydrochloride also increasing worm median survival. Compound ASW06 did not extend worm lifespan or improve worm survival at 10 µM concentration. Survival curve comparison were performed using Log-Rank (Mantel-Cox) test.

225

Supplementary 15 Experimental repeat #2 of C. elegans survival assay trialling lead G6PD-stabilising ASW small molecules. G6PD-stabiliser ASW03 improved worm survival at 50 µ concentration with no change to median survival observed with the 10 µM treatment. 10 µM ASW07 significantly improved worm survival, with small molecule ASW22 also significantly increasing worm median survival at both 10 µM and 50 µM dose. The G6PD inhibitor ASW25 did not change the survival of the animals. Survival curve comparison were performed using Log-Rank (Mantel-Cox) test.

226

Supplementary 4 Experimental repeat #3 of C. elegans survival assay trialling lead G6PD-stabilising ASW small molecules. G6PD-stabilisers ASW03, ASW08 and ASW21 improved worm survival when administered at 50 µM concentration with no significant changes observed to median survival observed following 10 µM treatments. Small molecule ASW22 also significantly increased worm median survival at both 10 µM and 50 µM dose, with a stronger improvement observed at the higher concentration. The G6PD inhibitor ASW25 did not significantly affect the survival of the animals. Survival curve comparison were performed using Log-Rank (Mantel-Cox) test.

227

Supplementary 5 Experimental repeat #4 of C. elegans survival assay trialling lead G6PD-stabilising ASW small molecules. G6PD-stabilisers ASW03 and ASW08 improved worm survival when administered at 50 µM concentration with no significant changes observed to median survival observed following 10 µM treatments. Small molecule ASW22 also significantly increased worm median survival at both 10 µM and 50 µM dose, with a stronger improvement observed at the higher concentration. Survival curve comparison were performed using Log-Rank (Mantel-Cox) test.

228

Supplementary 6 Experimental repeat #5 of C. elegans survival assay trialling lead G6PD-stabilising ASW small molecules. G6PD-stabilisers ASW03 improved worm survival when administered at 50 µM concentration with no significant changes observed to median survival observed following 10 µM treatment. Small molecules ASW08 and ASW22 also significantly increased worm median survival at both 10 µM and 50 µM dose, with a stronger improvement observed at the higher concentrations. Compound ASW06 trialled at 50 µM did not significantly affect the survival of the animals. Survival curve comparison were performed using Log-Rank (Mantel-Cox) test.

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