Isolation, Identification and Evaluation of New Anti-Prion Compounds from Australian Marine Invertebrates Using Novel Yeast-Prion Screening

Isolation, Identification and Evaluation of New Anti-prion Compounds from Australian Marine Invertebrates using Novel Yeast-prion Screening

Author Jennings, Laurence

Published 2018-11

Thesis Type Thesis (PhD Doctorate)

School School of Environment and Sc

DOI https://doi.org/10.25904/1912/722

Downloaded from http://hdl.handle.net/10072/382708

Griffith Research Online https://research-repository.griffith.edu.au

Isolation, Identification and Evaluation of New Anti-prion Compounds from Australian Marine Invertebrates using Novel Yeast-prion Screening

Written by Laurence Kane Jennings, BMarSc, BSc(Hons)

Griffith School of Environment & Science Griffith University (Gold Coast campus), Queensland, Australia

This thesis is submitted in fulfilment of the requirements of the degree of doctor of philosophy

November 2018

ABSTRACT

Prion diseases are fatal neurodegenerative diseases caused by the build-up of a misfolded form of the prion protein in the brain. These misfolded isoforms of the prion protein are infectious and capable of catalysing the transformation of the native protein into the same misfolded ‘prion’ form. Prion diseases include Creutzfeldt-Jakob disease (CJD) in humans, as well as scrapie and bovine spongiform encephalopathy (mad cow disease) in animals. Other neurodegenerative diseases caused by the build-up of misfolded proteins include Alzheimer’s disease, Parkinson’s disease, Huntington’s disease and Motor Neurone disease. An outbreak of CJD in the UK in the 1990s led to an extensive search for therapeutics to treat prion diseases. This resulted in the identification of a number of anti-prion compounds active in vitro, however, very few compounds active in vivo that could be used as drugs. Therefore, there is currently no curative therapeutic available for the treatment of these fatal diseases. The screening of large, diverse compound libraries has been considered an important strategy for the identification of novel anti-prion compounds. However, this has been hindered by a number of factors, including the limited of knowledge of prion mechanisms and the lack of suitable screening assays.

For this reason, we aimed to develop a new assay that could be used to screen chemically complex natural extracts for anti-prion activity. This assay was then used to identify novel anti-prion compounds from the marine environment, a large source of chemical and biological diversity previously untapped in the search for anti-prion compounds. The isolated anti-prion compounds were further evaluated and compared to previously identified compounds that lack sufficient activity in vivo.

Chapter One (Prion publication) describes the development of a new yeast-based anti- prion assay that can be used for screening naturally-derived extracts. This assay utilises prions of the yeast Saccharomyces cerevisiae as the basis for a simple colorimetric anti- prion screen. The yeast is white when infected with the yeast prion and red when the protein is in its non-infectious normal form. This assay was then used to screen 500 marine invertebrate derived extracts resulting in the identification of four extracts with activity. This chapter then describes the use of the anti-prion assay to target the isolation of the active compounds from the active extract from the Australian sponge, Suberea

II ianthelliformis. This resulted in the identification of three known bromotyrosine alkaloids with potent anti-prion activity.

Chapter Two describes the bioassay-guided isolation of active compounds from the anti-prion extract from the Australian ascidian, Polycarpa procera. Extensive purification and structural elucidation resulted in the identification of four new butenolide and two new propanone metabolites, the procerolides and procerones, respectively. These compounds exhibited potent anti-prion activity.

Chapter Three describes the bioassay-guided isolation of new anti-prion compounds from an active extract from the Australian ascidian, Didemnum sp. This resulted in the isolation and identification of a new set of sulfated poly-oxygenated sterol derivatives, the didemnisterols. These compounds were isolated in low yields but exhibit potent anti-prion activity. Further biological testing also showed that these compounds display binding to α-synuclein, another neurodegenerative disease causing misfolded protein, and inhibit its aggregation.

Chapter Four describes the bioassay-guided isolation of active compounds from an anti- prion extract from the Australian sponge, Dysidea sp. This resulted in the identification of four known poly-oxygenated sterol derivatives. These Dysidea-sterols displayed potent anti-prion activity in the yeast-based anti-prion assay.

Chapter Five describes the evaluation of the sixteen potent anti-prion natural products isolated in this study. We evaluate their anti-prion activity, physicochemical properties, neurotoxicity and ability to inhibit α-synuclein aggregation in vitro. This data was compared to that of previously identified anti-prion compounds and currently used CNS drugs for the selection of the most promising lead candidates. We suggest that the lead compounds from this study be further evaluated using structure-activity relationship studies to identify their important molecular fragments. This knowledge can then be used for the design of novel therapeutics to treat prion diseases.

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Statement of Originality

This work has not previously been submitted for a degree or diploma in any university. To the best of my knowledge and belief, the thesis contains no material previously published or written by another person except where due reference is made in the thesis itself.

(Signed)______

Laurence Kane Jennings Date:______

(Signed)______

Supervisor: Anthony R. Carroll Date:______

(Signed)______

Co-supervisor: Alan L. Munn Date:______

Acknowledgements

I would like to thank my supervisors, Prof. Anthony R. Carroll and Dr. Alan L. Munn who over the last 3-4 years have given me a great deal of support, patience and guidance. Without their continued support the outcomes of this PhD would not have been possible.

I also acknowledge the Carroll and Munn laboratory groups: Joshua Hayton, Leesa Klau, Fan Yang, Dayani Sarath Parakumge, Larissa Buedenbender, Guy Kleks, Tanja Voser, Joshua Porter, James Baxter, Darren Holland, Dale Prebble Ishtiaq Ahmed and Zain Akram for their support throughout my PhD. Particularly, Dr. J. Hayton, Mr. L. Robertson and Mr. I. Ahmed for their hours of help and support.

I would like to thank my lovely wife Shannon for her support and patience throughout my PhD. I also thank my family and close friends for their continued support over the last 3-4 years.

I would like to thank our collaborators whose support has made this project possible. We thank Prof. Marc Blondel, Dr. Cecile Voisset and Ms. Flavie Soubigou from CNRS in Roscoff, France for providing the yeast strains. We thank Dr. Wendy Loa-Kum- Cheung, and Mr. Jeremy Carrington for their technical assistance. We thank Prof. George D. Mellick and Mr. Mingming Xu for α-synuclein screening. We thank Assoc. Prof. Shailendra Anoopkumar-Dukie and Ms. Fleur McLeary for toxicity screening. We thank Dr. Santosh Rudrawar and Mr. Philip Ryan for the synthesis of compounds.

Additionally, I would like to thank the Environmental Futures Research Institute, Griffith Research Institute for Drug Discovery and Griffith School of Environment and Science for their financial support as well as equipment used. I would also like to acknowledge that this research was supported by an Australian Government Research Training Program Scholarship.

Table of Contents

ABSTRACT ...... II Statement of Originality ...... IV Acknowledgements ...... V List of Abbreveations ...... VIII List of Figures and Tables ...... X INTRODUCTION: The current understanding of prions and anti-prion therapeutic leads...... 1 Abstract ...... 2 Prions and their Properties...... 3 Methods of Screening for Anti-prion Compounds ...... 11 Anti-prion Therapeutic Leads ...... 15 Natural Product Drug Discovery ...... 33 Conclusion ...... 36 Aims, objectives and scope of the study ...... 37 References ...... 38 CHAPTER 1: Yeast-Based Screening of Natural Product Extracts Results in the Identification of Prion Inhibitors from a Marine Sponge...... 46 Statement of Contribution ...... 47 Prion publication: ...... 48 CHAPTER 2: Anti-prion Butenolides and Diphenylpropanones from the Australian Ascidian Polycarpa procera...... 59 Statement of Contribution ...... 60 Journal of Natural Products manuscript ...... 61 CHAPTER 3: New Anti-prion and α-synuclein Aggregation Inhibitory Sterols from the Australian Ascidian, Didemnum sp...... 85 Statement of Contribution ...... 86 Journal of Natural Products manuscript ...... 87 CHAPTER 4: Poly-oxygenated sterols from the sponge Dysidea sp. that exhibit potent inhibitory activity against yeast prions...... 106 Abstract ...... 107 Introduction ...... 108 Results and discussion ...... 109 VI

Experimental section ...... 125 References ...... 127 CHAPTER 5: Screening natural products for anti-prion activity and evaluation of isolated anti-prion compounds for selection as lead chemical scaffolds...... 128 Abstract ...... 129 Introduction ...... 130 Results and discussion ...... 132 Experimental section ...... 143 References ...... 145 CONCLUSIONS: Discovery of novel anti-prion compounds by screening natural product libraries using a yeast based assay: a success...... 147 APPENDIX ...... 150 Supplementary data - Chapter 1: ...... 151 Supplementary data - Chapter 2: ...... 166 Supplementary data - Chapter 3...... 192 Supplementary data - Chapter 4...... 211 Supplementary data - Chapter 5...... 218

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List of Common Abbreviations

PrP prion protein CJD Creutzfeldt-Jakob disease vCJD varient Creutzfeldt-Jakob disease FFI Fatal Familial Insomnia BSE bovine spongiform encephalopathy PrPC cellular prion protein (normal fold) PrPSc infectious prion protein (misfold) [PSI+] prion form of Sup35p [URE3] prion form of Ure2p Ade adenine Hsp heat shock protein GuHCl guanidine hydrochloride CNS central nervous system CSF cerebrospinal fluid BBB blood-brain barrier DMSO dimethylsufoxide PRAI poly(rybosylaminoimidazole) HTS high throughput screen UV ultra violet CID computer-imaging densitometry YPD yeast extract, peptone and dextrose media SD standard deveation HPLC high performance liquid chromatography RP reverse phase MS mass spectrometry LC-MS liquid chromatography with tandem mass spectrum Q-TOF quadrupole time of flight HRESIMS high resolution electrospray ionization mass spectrum LRESIMS low resolution electrospray ionization mass spectrum NMR nuclear magnetic resonance 1H NMR proton nuclear magnetic resonance 13C NMR carbon nuclear magnetic resonance 2D two dimensional

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COSY correlation spectroscopy HSQC heteronuclear single quantum coherence HMBC heteronuclear multiple bond correlations ROESY rotating-frame overhauser effect spectroscopy n JCH n bond hydrogen to carbon correlation (n = 2, 3, or 4) s singlet d doublet t triplet q quartet m multiplet br broad TDDFT time-dependent density functional theory IR infra-red PDA photo diode array detector

[α]D specific rotation Hz Hertz

DMSO-d6 deuterated dimethylsulfoxide TFA trifluoroacetic acid MeOH methanol ACN acetonitrile

C18 octadecyl bonded silica Da Dalton m/z mass to charge ratio Me methyl MTPA α-methoxy-α-(trifluoromethyl) acetic acid CD circular dichroism ECD electronic circular dichroism

EC50 concentration required to cure 50% of the yeast cells

List of Figures Figure I: The process of self-replication and amyloidosis by the prion protein. A spontaneous conformational 3 change in the PrPC leads to the PrPSc. This PrPSc catalyses the switching of the PrPC into PrPSc. The PrPSc aggregates into prion oligomers and then amyloid plaques. These oligomers and amyloid plaques then cause neurodegeneration and eventually death. Figure II: The [psi-] and [PSI+] yeast strains with the ade1-14 mutation grown on agar. This figure is adapted 6 from Tessier and Lindquist, 2009. Figure III: The role of Hsp chaperones in breaking amyloid plaques into prion-seeding fractions. 9 Figure IV: Different polyanionic glycans that have been found to have anti-prion activity: pentosan- 16 polysulfate (1), dextran sulfate (2), the carrageenans (3), and heparin sulfate (4). NOTE: R1, R2 and R3 can be - OH or OSO3 . Figure V: Different azo dyes and related compounds that have been found to possess anti-prion activity: 18 congo red (5), thioflavin-S (6), chicago sky blue (7), curcumin (8), suramin (9), and dapsone (10).

Figure VI: Different tetrapyrrole compounds that have been found to have anti-prion activity: 19 duteroporphyrins (11), tetraphenyl porphines (12), and phthalocyanines (13). Figure VII: Different low weight chaperone-like reagents that have been found to exhibit anti-prion activity: 20 DMSO (14), glycerol (15), and TMAO (16). Figure VIII: Some of the polyphenolic compounds that have been found to have anti-prion activity: tannic 21 acid (17), katacine (18), epicatechin monogallate (19), epigallocatechin 3,5-digallate (20), 2,3,5,7,3’,4’- pentahydroxyflavan (21), and baicalein (22). Figure IX: Some of the most active phenothiazine, acridine and quinoline derivatives that have been found to 22 have anti-prion activity: quinacrine (23), chloroquine (24), chlorpromazine (25), quinine (26), 6-amino-8- chloro-phenanthridine (27), tacrine (28), bisquinoline (29) and bisacridine (30). Figure X: Different aromatic PrP-binding compounds that have been found to have anti-prion activity: 23 benzylidene-benzohydrazide 293GO2 (31), diphenyl-pyrazole anle138b (32), guanabenz (33), and indole- glyoxylamide (34). Figure XI: Different PrPC binding compounds that have been found to have anti-prion activity discovered 24 using virtual screens: dicarbonitrile Cp60 (35), methoxybenzene sulfonohydrazide Cp62 (36), GN8 (37), and diaryl-2-aminthiozole (38). Figure XII: Different dendrimers that have been found to have anti-prion activity: polyamidoamide 1.0 (39), 26 polypropyleneimine (40), and phosphorus containing polyamine dendrimer 1.0 (41). Figure XIII: The anti-prion tyrosine kinase inhibitor imatinib mesylate (42). 27 Figure XIV: Different tetracycline compounds that have been found to have anti-prion activity: 28 anthracycline 4’-iodo-4’-deoxy-doxorubicin (43), tetracycline (44), and doxycycline (45).

Figure XV: Different polyene compounds that have been found to have anti-prion activity: amphotericin B 30 (46), filipin (47), and MS-8209 (48).

Figure XVI: Other cholesterol lowering drugs that have anti-prion activity: lovastatin (49), simvastatin (50), 31 squalastatin (51) and verapamil (52). Figure XVII: Metal-chelating compounds that have anti-prion activity: D-penicillamine (53), clioquinol (54), 32 cimetidine (55), neocuproine (56), bathocuproine (57) and 2,2’-biquinoline (58). Figure XVIII: (a) A pie chart showing the proportion of all new drugs over the last 30 years that have been 34 developed from various sources. This figure is adapted from Newman and Craig, (2016).209 (b) Common drugs that have been developed from natural products: morphine (59), penicillin G (60), taxol (61) and artemisinin (62). Figure XIX: A pie chart displaying the origin of the anti-prion compounds that have been identified. 35 Figure XX: Principal component analysis of structural features of the compound libraries. This figure is 35 reproduced from Feher et al. (2003). a) the diversity of synthetic combinatorial compounds, b) the diversity of natural products, and c) the diversity of drugs in current clinical use. Figure 1-1: A schematic diagram giving an overview of the large-scale screen (from left to right). A liquid- 50 phase micro-culture assay is performed on each sample in a library of marine extracts and the color change of the yeast cultures from white to red indicates antiprion activity. The marine invertebrate containing the compounds with anti-prion activity, the sponge Suberea ianthelliformis (pictured), underwent chemical analysis to identify the anti-prion compounds. Figure 1-2: A comparison of the accuracy and precision of the three methods of quantitating PRAI a. 50 absorbance, b. fluorescence and c. CID. d. the scanned image of the micro-titer plate. The top four wells are 100% [psi−] cultures, the middle four wells are 50% [psi−]/50% [PSI+] cultures and the bottom four wells are X

100% [PSI+] cultures (see the experimental section for [psi−] control cultures). Figure 1-3: The optimization of the concentration of yeast extract in YPD medium and the yeast cell density 52 of inoculates. a. the color intensity of prion-cured and infected [PSI+] cultures with different concentrations of yeast extract in the YPD medium. b. color intensity of micro-titer plate wells after cultures reached stationary phase relative to cell density at inoculation. Color intensity in both graphs is shown relative to the [PSI+] and [psi−] control cultures in graph a (see experimental section) and was measured using absorbance at 540 nm. Figure 1-4: Experimental results for the validation of the screen with known anti-prion compounds. a. the 52 toxicity of DMSO for the primary STRg6 [PSI+] yeast cells. Inhibition was measured through colony counting and was relative to a 0% (inoculated with no DMSO) and 100% (no cell inoculation) growth inhibition. b. percentage curing by guanabenz as a function of the concentration of GuHCl. Each curve represents the addition of a different sub-effective concentration of GuHCl to the growth medium. The color intensity was measured using CID and was relative to controls ([PSI+] had addition of DMSO and [psi-] had addition of 2mM GuHCl). Figure 1-5: Chemical structures of the natural products identified in this study and respective screening 54 results. a. Flatbed scanned image of the wells of the micro-titer plate. This displays the change in color of the STRg6 yeast cultures when grown in the presence of the three compounds (one replicate). b. Dose-response curves for the anti-prion activity against [PSI+] of the three active compounds. The dotted black line displays the anti-prion activity of guanabenz in the assay, indicating the baseline for potent anti-prion activity. The color intensity was relative to the controls (200 μM guanabenz for the positive) and was measured using CID. Figure 2-1: New butenolides: the procerolides (1 - 4) and new diphenylpropanones: the procerones (5 - 6), 64 isolated from P. procera. Figure 2-2: (a) Key COSY and HMBC correlations for 1. (b) Comparison of the experimental ECD spectrum 68 of 1 and the calculated spectrum of the (S)- and (R)- enantiomers of 1 at the B3LYP/6-31G(d) level. Figure 2-3: (a) The proposed biosynthetic pathway of the procerolides and then to the procerones. (b) Key 72 COSY and HMBC correlations for 5. (c) Δδ values [Δδ (in ppm) = δS – δR] obtained for (S)- and (R)-MTPA esters of 5. (d) Comparison of the experimental ECD spectrum of 5 and the calculated spectrum of the (S)- and (R)- enantiomers of 5 at the B3LYP/6-31G(d) level. Figure 2-4: The proposed tautomeric shift within the procerolides, (a), and the procerones, (b), under basic 74 conditions with Δδ values of protons [Δδ (in ppm) = δacidic – δbasic]. (c) The observed bathochromic that accompanied the tautomeric shift of both 1 and 5. Figure 3-1: New anti-prion sterols, the didemnisterols (1-3), isolated from Didemnum sp. 90 Figure 3-2: (a) Selected COSY and HMBC correlations of 1-3. (b) Chem3D energy minimised structure of 1 94 with selected ROESY correlations. Figure 3-3: Previously isolated sulfated steroids from ascidians: Ascidia-SAAF (4), Ciona-SAAF (5), 96 phallusiasterols A-C (6-8). Figure 3-4: MS binding assay spectrums: (a) The mass spectrum of α-synuclein alone, with arrows 98 identifying the charge state of each peak. (b) The mass spectrum of α-synuclein after incubation with didemnisterol A, with arrows identifying different the additional peaks associated with an α-synuclein- didemnisterol complex. Figure 4-1: Isolation diagram of the anti-prion poly-oxygenated dysidea-sterols (1-4). 109 Figure 4-2: Structure of Dysidea-sterol A-11-acetate (1) 110 Figure 4-3: 1H NMR spectrum of Dysidea-sterol A-11-acetate (1) and Dysidea-sterol B-11-acetate (2) in 1:1 111 mixture in DMSO-d6 Figure 4-4: Key COSY and HMBC correlations for the elucidation of the structure of 1 113 Figure 4-5: Key ROESY correlations used to assign the relative configuration of 1 114 Figure 4-6: Structure of Dysidea-sterol B-11-acetate (2) 115 Figure 4-7: Key COSY and HMBC correlations of the side chain of 2 116 Figure 4-8: Structure of Dysidea-sterol A-11,19-diacetate (3) 117 Figure 4-9: 1H NMR spectrum of a mixture of Dysidea-sterol A-11,19-diacetate (3) and Dysidea-sterol B- 118 11,19-diacetate (4) in DMSO-d6 Figure 4-10: Configuration of the diacetate Dysidea-sterol A-11,19-diacetate (3) 119 Figure 4-11: Structure of Dysidea-sterol B-11,19-diacetate (4) 120 Figure 4-12: Acetylcholinesterase inhibiting natural products isolated from the Dysidea genus 122 Figure 4-13: The oxygenated C-19 sterol derivatives isolated from the Dysidea genus, 9/11-epoxy sterols (8), 123 11/19-ether sterols (9), herbasterols (10) and 5β-dysidea-sterols (11). XI

Figure 5-1: The anti-prion compounds identified in the current bio-discovery effort. 130 Figure 5-2: Anti-prion compounds selected for comparison to anti-prion activity of the isolated natural 132 products: quinacrine (17), guanabenz (18), anle138b (19), and 6-aminophenanthridine (20). Figure 5-3: 3D histogram of the ClogP vs. tPSA values for (a) 120 currently used CNS drugs, (b) the natural 136 products isolated in this study (NP, 1-16), and (c) the other anti-prion compounds described in the introduction. Figure 5-4: The cell viability of SHSY-5Y neuroblastoma cells following treatment with the anti-prion 137 natural products at three concentrations. Additionally, guanabenz and quinacrine were included for comparison. The three controls from left to right are media (No cells), DMSO vehicle control (with cells) and no treatment control (with cells). Shown are the bars representing mean + standard error bars for each treatment done in triplicate. Figure 5-5: A preliminary screen of the anti-prion natural products and known anti-prion compounds for 139 inhibition of α-syn aggregation in vitro using ThT fluorescence. The controls are the 72 hour untreated negative control and the epigallocatechin gallate (EGCG) positive control. Shown are the bars representing the ThT fluorescence of samples that correlates to the α-syn aggregation. Figure 5-6: A further screen of procerolides A-D (4-7) and procerone A (8) for inhibition of α-syn 140 aggregation in vitro using ThT fluorescence. The controls are the 72 hour untreated negative control and the epigallocatechin gallate (EGCG) positive control. Shown are the bars representing mean of the ThT fluorescence + standard error bars for each treatment done in triplicate. Figure 5-7: A further screen of the poly-oxygenated sterols: didemnisterol A (10) and Dysidea-sterol-11- 141 acetate mixture (13/14) for inhibition of α-syn aggregation in vitro using ThT fluorescence. The controls are the 72 hour untreated negative control and the epigallocatechin gallate (EGCG) positive control. Shown are the bars representing mean of the ThT fluorescence + standard error bars for each treatment done in triplicate.

List of Tables Table I: Table of common amyloid based prion proteins in yeast. NOTE: this table is adapted from Liebman 7 and Chernoff, 2012.

Table 2-1: NMR Spectroscopic Data for Procerolides A - D (1 - 4) in DMSO-d6. 64

Table 2-2: NMR Spectroscopic Data for Procerones A - B (5 - 6) in DMSO-d6. 69 Table 2-3: Anti-prion activity of the procerolides (1-4) and the procerones (5-6). 75

Table 3-1: NMR spectroscopic data for didemnisterol A-C (1-3) in DMSO-d6. 91 Table 3-2: Anti-prion and α-synuclein aggregation inhibitory activity of the didemnisterols (1-3) compared to 97 other known anti-prion and α-synuclein aggregation inhibiting compounds.

Table 4-1: NMR data for Dysidea-sterol A-11-acetate (1) in DMSO-d6. 110

Table 4-2: NMR data for Dysidea-sterol B-11-acetate (2) in DMSO-d6. 115

Table 4-3: NMR data for Dysidea-sterol A-11,19-diacetate (3) in DMSO-d6. 117

Table 4-4: NMR data for Dysidea-sterol B-11,19-diacetate (4) in DMSO-d6. 120 Table 4-5: Anti-prion activity of the poly-oxygenated Dysidea-sterols. 123 Table 5-1: The physicochemical properties and ligand efficiency of the identified anti-prion natural products. 134

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Acknowledgement of co-authored publications as part of the thesis

This thesis contains co-authored publish and unpublish (in preparation) research papers in Chapters 1-3. My contribution to each co-authored paper is outlined at the front of the relevant chapter. The bibliographic details (if published or accepted for publication)/status (if prepared or submitted for publication) for these papers including all authors, are:

Chapter 1: Jennings, L. K.; Ahmed, I.; Munn, A. L.; Carroll, A. R.; “Yeast-Based Screening of Natural Product Extracts Results in the Identification of Prion Inhibitors from a Marine Sponge”. Prion 2018, 234-244.

Chapter 2: Jennings, L. K.; Robertson, L. P.; Rudolph, K. E.; Munn, A. L.; Carroll, A. R.; Anti-prion Butenolides and Diphenylpropanones from the Australian Ascidian Polycarpa procera. J. Nat. Prod. In preparation for submission

Chapter 3: Jennings, L. K.; Prebble, D. W.; Xu, M.; Munn, A. L.; Mellick, G. D.; Carroll, A. R. New Anti-prion and α-synuclein Aggregation Inhibitory Sterols from the Australian Ascidian, Didemnum sp. J. Nat. Prod. In preparation for submission

(Signed)______

Laurence Kane Jennings Date:______

(Signed)______

Supervisor: Anthony R. Carroll Date:______

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INTRODUCTION:

The current understanding of prions and anti-prion therapeutic leads

ABSTRACT Prion diseases are fatal, neurodegenerative diseases caused by the smallest infectious particle, the prion protein (PrP). Prion diseases include Creutzfeldt-Jakob disease (CJD) in humans, as well as scrapie and bovine spongiform encephalopathy in animals. More recent research has displayed strong links between the PrP and its mechanisms with other neurodegenerative diseases including Alzheimer’s disease, Parkinson’s disease, Huntington’s disease and Amyotrophic Lateral Sclerosis. With the outbreak of CJD in the UK in the 1990s there was a great interest in the search for therapeutics to treat these diseases. Here we review the current anti-prion therapeutic leads that have been identified. While a number of in vitro active anti-prion compounds have been identified these compounds have failed to be effective in vivo. This has been primarily due to poor permeation through the blood-brain barrier (BBB) and toxicity. As such, there is currently a low chemical diversity of compounds that can successfully pass the BBB with limited toxicity, of which none have been successful when used after onset of disease symptoms. We conclude that there is a need for a greater number of novel anti- prion compounds and that the targeting of novel natural extract libraries may fill this need.

PRIONS AND THEIR PROPERTIES The term ‘prion’ is derived from ‘proteinaceous infection particle’. Prions are misfolded infectious isoforms of a protein that are able to transform the native protein isoform into the same infectious isoform. Prion diseases are a rare group of infectious neurodegenerative disorders that are caused by a modified isoform of the prion protein (PrP).1 Prion diseases include variable Creutzfeldt-Jakob disease (vCJD), Gerstmann- Straussler-Scheinker syndrome (GSS), Fatal Familial insomnia (FFI), kuru in humans, and bovine spongiform encephalopathy (BSE), scrapie and chronic wasting disease (CWD) in animals.2 Currently there is no effective treatment for these neurodegenerative disorders. 3, 4

Figure I: The process of self-replication and amyloidosis by the prion protein. A spontaneous conformational change in the PrPC leads to the PrPSc. This PrPSc catalyses the switching of the PrPC into PrPSc. The PrPSc aggregates into prion oligomers and then amyloid plaques. These oligomers and amyloid plaques then cause neurodegeneration and eventually death. There are two forms of the prion protein; the normal form (PrPC) and the pathological form (PrPSc). The conformation of PrPC possesses a large proportion of α-helices and unstructured amino acids, whilst possessing a small proportion of -sheets. In this form the protein is soluble, globular and sensitive to proteinases as well as being able to perform its general biochemical function. When the conformation of PrPC switches to PrPSc its quaternary (3D) structure significantly changes such that it possesses a larger portion of β-sheets and as a consequence it forms large insoluble aggregates or amyloid plaques that are resistant to digestion by proteinases.5 The PrPSc form of the protein is also resistant to various chemical and physical denaturation and degradation processes and so the aberrant protein accumulates in diseased tissues. The PrPSc effectively catalyses the transformation of PrPC to the pathogenic PrPSc form allowing the PrPSc to self-replicate.6 These prions then associate to form toxic oligomers and then insoluble 3

prion amyloids in a process known as the amyloid cascade. The amyloid plaques are then split into seeding fractions that cause the accelerated recruitment of the PrPC and prevent the protein from performing its normal biochemical function.2 Studies have shown that the amyloid cascade caused by misfolded proteins is associated with over 30 other human diseases including Alzheimer’s, Parkinson’s, and Huntington’s diseases.3

Mammalian Prions In mammals, prion diseases are referred to as transmissible spongiform encephalopathies (TSE). This is due to the typical symptoms common amongst these diseases, including the transmissible nature of the diseases and neurodegeneration.2 Prion proteins were first discovered in sheep inflicted with a fatal disease that caused the sheep to scratch themselves to death. This disease called Scrapie was found to be a neurodegenerative disorder caused by the build-up of prion proteins in the sheep’s brain.7, 8 The scrapie prion is the most studied of the known mammalian prions, however it has been hypothesised that there are potentially many more mammalian proteins susceptible to prion-like switching that remain unknown.9

The inability of prions to transmit between differing species is known as the species barrier phenomenon. Many studies have shown that there is a biochemical barrier that stops most interspecies prion infections. However it has been shown that if the domains of these proteins are highly similar then infectious proteins can be transmitted between species.10, 11 This was the case for the prion involved in BSE since the bovine prion protein was able to change the conformation of the human equivalent protein causing vCJD.10 However, other prions such as the scrapie prion have not been shown to cross the species barrier due to great differences in the equivalent protein.11

Prions were first identified in humans in Papua New Guinea with patients suffering from a neurodegenerative disorder called Kuru which is associated with ritualistic cannibalism. Interestingly, other prion diseases in cows and sheep have also been shown to be associated with cannibalism.7 BSE is an example of a prion disease that originated from feeding cattle offal from beef abattoirs. BSE is similar to human prion diseases and causes neurodegeneration in the brains of cattle. The association between cannibalism and prion diseases, as well as the mechanisms that initially cause prions have not been studied in detail. Environmental and genetic stress factors as well as age have all been associated with the initial misfolding of the prion protein.12 It is known, however, that the disease can be transmitted via oral routes due to the resistant nature of

the infectious prion protein. This was seen in the outbreak of CJD in the UK during the 1990s when the infectious bovine prion protein was transmitted through the eating of BSE-infected beef products.13

In humans, the PrPC is a copper-binding glycosylphosphatidylinositol-anchored protein on the cell surface. The exact biological functions of this protein are unknown, however, recent studies have suggested that it has a number of important roles in the central nervous system.14 It has been hypothesized that PrPC is switched to the infectious prion form in lipid-rich micro domains at the surface of the cell.15 The PrPSc then associates to form the oligomers and then amyloid fibrils which in mammals cause cell death and eventually leads to organ failure. The accumulation of these amyloids fibrils in the central nervous system (CNS) and in the spleen is the main diagnostic feature of prion diseases.16 While it was originally thought that the toxicity was due to the amyloid fibrils, recent studies have suggested that the neurotoxicity is primarily caused by the prion oligomers.17

To date, research on mammalian prions has been slow due to difficulties associated with studying them. This has mainly been due to the fact that prions are highly infectious and can only be studied in high security laboratories.18 As a consequence, a number of researchers have looked for alternative tools to understand human prions and prions found in yeasts have been used as a model system.19

Yeast Prions Prions in the yeast Saccharomyces cerevisiae were first proposed in 1994 by Reed Wickner. It was proposed that two previously non-heritable phenotypes [URE3] and [PSI+] were caused by the prion forms of the Sup35 and Ure2 proteins, respectively.20, 21 These phenotypes are characterised by the media-specific growth of colonies dependent on whether Sup35p and Ure2p are in their respective prion forms. The uninfected phenotypes, [psi-] and [ure-o], are characterised by a lack of growth on adenine deficient, and ureidosuccinic acid containing media, respectively.22 These phenotypic changes occur because normal Sup35p and Ure2p are not able to perform their normal biochemical function after they are converted to their infectious prion form. Since this discovery, several additional prion-like proteins have been discovered in yeast (Table I- 1).23

These prion-infected strains have since been genetically modified to exhibit a phenotypical colour change that can be used to more easily distinguish between the non- 5

infected and infected cells.18, 22 In these [PSI+] strains the colouration of the yeast is caused by the prion form of the Sup35p.11 The Sup35p is an important factor for translation termination during synthesis of the Ade1p. The functional Ade1p yeast protein is important for the synthesis of adenine to allow growth of the cells on adenine- deficient minimal medium (SD-Ade).22, 24 In the genetically modified [PSI+] strains a premature stop codon has been placed in the ADE1 gene so that ade1-14 mRNA is produced. In [psi-] cells where Sup35p is in its normal soluble (non-prion) form it performs its normal biochemical function and the translation of the ade1-14 allele is terminated at the premature stop codon causing the formation of the truncated Ade1p. This truncated Ade1p is unable to function in the synthesis of adenine at a late stage in its biosynthesis and this leads to the build-up of the red coloured adenine precursor poly-(ribosylaminoimidazole) (PRAI) in the cells.21, 25 In [PSI+] cells the Sup35p in its prion form, aggregates and does not mediate this translation termination. This leads to the ribosomes becoming less efficient at reading the premature stop codon in the ade1- 14 mRNA.21 Because of this the cells produce full length functional Ade1p enzyme leading to the formation of white cells. Thus, the yeast infected with the [PSI+] prion are white and un-infected [psi-] yeast is red.22, 24

Figure II: The [psi-] and [PSI+] yeast strains with the ade1-14 mutation grown on agar. This figure is adapted from Tessier and Lindquist, 2009.26 The [URE3] colouration of the yeast is caused by the prion form of the Ure2p. Ure2p is important for repression of nitrogen metabolism genes.20, 27 The DAL5 gene is one of these nitrogen metabolism genes that is not transcribed when the Ure2p is active due to transcription repression, however when the Ure2p is in its prion form it aggregates, loses function and the DAL5 gene can be transcribed.22, 28 In the [URE3] yeast reporter strain the DAL5 gene has been replaced with the ADE2 gene under the DAL5 promoter. Transcription of a functional Ade2p is essential for the biosynthesis of adenine. In [ure-

o] yeast cells ADE2 is not transcribed, adenine biosynthesis is blocked and a build-up of the same red adenine precursor PRAI occurs, leading to the formation of red colonies. In the [URE3] yeast cells ADE2 is actively transcribed only when the Ure2p is in its prion form and this leads to the biosynthesis of adenine and the formation of white colonies.29 Therefore, like yeast infected with the Sup35p [PSI+] prion, yeast infected with the Ure2p [URE3] prion produce white cells and those that are not infected produce red cells.27, 29

Sup35p has a mammalian equivalent, the eRF3 protein, which is responsible for translation termination in mammals. However, Ure2p has no mammalian equivalent.30 There are several other yeast prions that have been identified that can be present in either a normal soluble form or a transmissible amyloid form. Many of the yeast proteins have a mammalian equivalent protein with high homology.23

Table I: Table of common amyloid based prion proteins in yeast. NOTE: this table is adapted from Liebman and Chernoff, 2012.22 Prion Protein Normal function Mammalian related. [PSI+] Sup35 Translation termination eRF3 [URE3] Ure2 Nitrogen regulation None [PIN+]/[RNQ+] Rnq1 Protein template factor Unknown [SWI+] Swi1 Chromatin-remodelling Arid1A transcription factor [OCT+] Cyc8 Transcriptional repressor Unknown [MOT3] Mot3 Nuclear transcription Plasma proteins, factor Predicted membrane proteins, transporters [ISP+] Sfp1 Global transcriptional Akt1 regulator

Whilst in mammals it is unknown why these proteins misfold, in yeast theories have been developed to explain this phenomenon. It has been theorised that yeast prions add a new mechanism for genetic variation within a population that can potentially give the yeast an evolutionary advantage.31 In more recent years research on the marine mollusc Aplysia californica, has shown that prions may also be an important part of the mechanism of long term information storage (memory) in their brain.32 These studies indicate that prions and related proteins may have important biological roles in mammals and that the current understanding of prions is currently very limited.

Prion variants and propagation Prion variants are different protein isoforms that confer different phenotypic characteristics.19 Prion variants were originally observed in mouse scrapie with differing replicates of the same prion strain displayed differing incubation times.23 The different

infectious characteristics in mammalian prion variants include differing incubation times (time until onset of disease), distributions of lesion, and host ranges (the range of cells the prion can infect).11, 33 These variations in the prion function are uniquely associated with variation in the amyloids such as structural differences.33 The structure of the prion amyloid is an irregular parallel β-sheet structure34 which is maintained mainly by hydrogen bonds between glutamine (Q) and asparagine (N). The alteration of the structure of amyloid monomers through amyloid breakages and templating can create new self-propagating amyloid structures that can become new prion variants. However the amyloid structures are unknown for different prion variants.22 By analogy, it has been found that yeast prions also exhibit different prion variants as seen in mammals due to similar cellular mechanisms. This shows that yeast are a good model for the study of prion variation and propagation.22 In yeast these variations include their stability, the intensity of the phenotype and the susceptibility of the prion form to the depletion or overexpression of the different chaperones.31

In [PSI+] yeast different variants display differing phenotype intensities (intensities of red colouration of cells). These differences are associated with different ratios of aggregated to non-aggregated Sup35p. This variation in the aggregation of Sup35p causes differing levels of translation of Ade1p and thus red colouration. Different [PSI+] prion variants were thus termed strong and weak,35, 36 dependent on the aggregation characteristics of Sup35p. Strong [PSI+] variants typically have a greater number of small-sized prion seeding aggregates allowing a greater chance for the conversion of the protein to the prion.31 Weak [PSI+] variants typically have a smaller number of large- sized prion aggregates and thus have a lower chance for the conversion of the protein to the prion.25, 31 This leads to less misfolded prions in weak variants and greater translation of the ade1-14 allele. Different variants of other yeast prions have also been observed having differing phenotypic characteristics based on the aggregation.22

The propagation of yeast prions has been shown to depend on chaperone machinery. Different Hsps (heat-shock proteins) are the major chaperones involved in the maintenance and propagation of the [PSI+] prion.37 This was discovered from the observation of aggregation variation and curing of the [PSI+] prion with depletion or over-expression of various Hsps. As well as these Hsps having an effect on [PSI+] they are also important for the maintenance of many other amyloid-based yeast and mammalian prions.38

The Hsp chaperones are used within the cell to effect the disaggregation and refolding of damaged individual proteins within an aggregate. For prions, the more highly ordered nature of their aggregates alters the interaction between the protein aggregate and the Hsp chaperones. This causes the Hsp chaperones to fragment the aggregates multiplying the aggregates into prion-seeds and thus causing prion propagation.22, 38 The efficiency of Hsp chaperones in the fragmentation of aggregates leads to different prion variants and thus different rates of infection.31 Aggregates of strong prion variants are readily fragmented to produce a larger number of aggregate seeds. Aggregates of weak prion variants are not fragmented as efficiently, thus causing fewer aggregate seeds.22, 34

Figure III: The role of Hsp chaperones in breaking amyloid plaques into prion-seeding fractions. The main Hsp required for the propagation of yeast-prions is Hsp104p, a hexameric ring-shaped AAA+ protein (ATPases Associated with diverse cellular Activities) that re- solubilizes the aggregated prion amyloids by forcing the amyloids through a central channel.39 Hsp104p is thought to be the most important chaperone in the propagation of yeast prions as the depletion of Hsp104p has been shown to cure all yeast amyloid prions excluding [ISP+]. The overexpression of Hsp104p in [PSI+] yeast cells has been shown to switch these cell to [psi+] cells.40

Similarly to yeast, in mammals Hsps are also important for the propagation of prions. Hsp104p is the major chaperone for prion propagation in yeast, however this protein is not conserved in multicellular organisms. While Hsp104p is not conserved in mammalian cells there are other similar chaperones and other Hsps that are conserved in mammalian cells that are important for the propagation of mammalian prions.22

Prion Domain From studying amino acid sequences common to yeast prions, a prion domain (PrD) has been identified. The PrD is a stretch of amino acids that are needed for the formation

and propagation of prions. The main characteristic of yeast PrDs is that the peptide sequences are rich in the polar uncharged amino acids glutamine (Q) and asparagine (N). The PrD has been shown to generally be richer in N than Q,41 however different ratios of the two residues can greatly affect the formation and propagation of prions. When the peptide sequence becomes enriched with more N than Q then the protein will readily form prion amyloids that are mildly toxic. However when this sequence becomes enriched with more Q than N then the protein will form soluble non-prion oligomers that are highly toxic. It is thought that the QN-rich peptide sequence is important for the aggregation and amyloid cascade of prions.42 Yeast and mammalian prions also contain multiple imperfect oligopeptide repeat (OR) sequences in addition to the QN-rich sequence. It is thought that these OR sequences aid in the propagation of the prions, however these OR sequences are not necessary for protein aggregation. It has therefore been hypothesized that the OR sequences are important for the self- replication of prions.22 In Saccharomyces cerevisiae there are almost 200 proteins with these QN-rich peptide sequences,41 and well over 200 proteins in humans that have been found to contain amino acid sequences similar to the PrD.32

Other misfolding protein and amyloid disorders Research has implicated misfolded proteins and amyloid formation in many other neurodegenerative disorders, including Alzheimer’s disease, Parkinson’s disease, Huntington’s disease and amyotrophic lateral sclerosis.18, 43, 44 These neurodegenerative diseases have in common the process of accumulation of amyloids from misfolded protiens.45 Other misfolded proteins involved in neurodegenerative diseases include tau and β-amyloid associated with Alzheimer’s disease,46 α-synuclein associated with Parkinson’s disease and multiple systems atrophy,47 FUS, TDP-43 and SOD1 proteins associated with Amyotrophic lateral sclerosis48 and Huntingtin protein associated with Huntington’s disease.49 While these misfolded proteins all have differing routes to the misfolded structure of the protein, once misfolded they all follow the same amyloid cascade that leads to neurotoxicity and neural apoptosis. As well as this, many important cellular mechanisms of prion diseases, including chaperone proteins and processes, are involved in the maintenance and toxicity of these other misfolded proteins.44 Other similarities include the requirement for a number of cellular components for maintenance and propagation of prions that are current drug targets for other neurodegenerative disorders. These components include acetylcholinesterase (AChE), cholesterol and redox-active metals. It has been shown that AChE affects the 10

aggregation of PrP and that the inhibition of AChE reduced the build-up of PrP amyloids.50 An increase in the levels of reactive-oxygen induced by copper and iron is also associated with an increase in the pathogenesis of the PrP in a similar way to what has been seen in the above diseases.51

Until recently, prion proteins and their amyloid forms were thought to only be associated with neurodegenerative disorders. However, recent research has shown that these prion proteins may play important roles in many other diseases, including many cancers, microbial infections, diabetes and autoimmune disorders.3 These diseases have been associated with prions due to sharing similar misfolded proteins and/or processes, or due to the over-expression of the human prion protein in the diseased cells.52 Studies have shown a link between prion-like protein aggregation and pathogenic microbes such as bacteria, fungal and protozoan parasites. Significant disease producing microbes include drug-resistant bacteria and Plasmodium sp.53 Hypotheses about how prions are linked to pathogenic micro-organisms include their interaction with a range of efflux transporter proteins and their generation of surface aggregates producing a protective barrier on the surface of the microbial cell.54, 55 Cancer and diabetes have been linked to prion diseases due to similar mechanisms of transmission and propagation as well as the over-expression of PrPC.16, 52

Whilst a number of diseases have been associated with prions, the involvement of prions in pathogenesis is still poorly understood. The research to discover compounds to cure prion infection could potentially help to unravel mechanisms associated with prion formation, aggregation and propagation and these discoveries could help to find new targets for the treatment of a diverse range of disease.

METHODS OF SCREENING FOR ANTI-PRION COMPOUNDS A number of prion disease models have been developed to allow screening for novel drug leads to be conducted. These include in vitro mammalian cell line and cell-free models, as well as in vivo fungal and animal prion models.56

Mammalian Cell line Models The majority of screening to discover compounds to inhibit prion infections has been done using mammalian cell-based assays. These assays mostly use different variants of

the sheep scrapie prion.57 However, other cell lines have also been developed to investigate other prion diseases. These include the variants of CJD, CWD and BSE.58

There are only a few different cell lines that have been identified that can be infected with scrapie prions. This is mainly due to the difficulty of propagating sheep prions in non-sheep cells.59 The most widely used cell lines (due to their ability to stably propagate prions) are mouse neuroblastoma cells, with the neuro-2A cells (N2a) being the most popular. These N2a cells have been shown to propagate a number of different prion variants, including Chandler, RML, 139A, 22L, C506, Fukuoka-1, and FU CJD. These scrapie-infected cell lines, better known as ScN2a, have been the main cell model used for the research and development of anti-prion therapeutic agents.60 Other neuronal cell lines that have been developed to stably propagate prions include mouse hypothalamic cells (GT1), hamster brain cells (HaB) and rat pheochromocytoma- derived line (PC12). There have also been a number of non-neuronal cell lines that have been developed. These include mouse brain cells from mesodermal origin (SMB), mouse fibroblast cells (L fibroblast), mouse NS1 cells fused with spleen cells (NS1) and rat glial cells. These cell lines are useful for the screening of compounds against PrP that have a high amount of prion conversion in non-neuronal cells.61 The ScN2a line has also proven to be useful to study the biology and mechanisms of prion replication and Sc propagation, for example it has been used to discover the location at which PrP forms in cells.62

A number of different compounds have been found to be active against prions using mammalian cell-based assays.57 However, while a number of compounds have shown activity in mammalian cell models, these models have largely failed to identify compounds with good in vivo activity.61 Recent studies have suggested that these models are bad predictors of in vivo activity due to the use of immortalised cells that are difficult to maintain in a prion-infected state. Moreover, prions harboured in these cells decrease in infectivity over time and have a much faster turnover rate than prions harboured in brain cells.58 While mammalian cell models offer a good first screen to confirm cellular activity against prions they may not be useful for initial screening of large compound libraries due to a large number of false positive results.

Cell-free Models There are three cell-free assays that are commonly used to discover anti-prion compounds: cell-free conversion inhibition assays, cell-free aggregation inhibition

assays and, more recently, PrP binding assays. All assays use only the purified PrPC and PrPSc.

The cell-free conversion and aggregation models use purified PrPC that is seeded with PrPSc. PrPSc effectively catalyses the conversion of the PrPC into the infectious PrPSc which then aggregates.63 The conversion from the PrPC to the infectious PrPSc or the aggregation of PrPSc can then be examined in the presence of compounds. Percent conversion is relatively easy to obtain, as the PrPSc is proteinase-resistant whereas the PrPC is not. The amyloid plaques can also be easily quantified with the use of fluorescence dyes. These are useful as they are a cheap, quick and highly reproducible assay for use in screening new compounds.63, 64 These methods are quite often used as secondary screens to obtain information on whether compounds are having their effect directly or indirectly on PrP switching.65

PrP binding assays are commonly used to find the coefficient of binding of compounds to PrP. However, recently, since the three dimensional structure of both forms of the PrP has been determined, these assays have become popular to identify and design compounds that bind strongly to PrP.66, 67 The first stage of this screening is typically computerised and focuses on the physical properties of the molecules to identify if there is expected to be binding.68 This is done by creating an interaction pharmacophore model based on where previous prion conversion inhibitors bind. Large libraries of compounds can then be virtually screened to identify potential compounds that can bind to these regions.68-70 Compound binding is then confirmed using experimental affinity and surface plasmon resonance (SPR) assays after the virtual screen.68

Cell-free assays are useful for identifying compounds that bind directly to PrP, thereby inhibiting the conversion and/or the aggregation. However, with a number of other cellular chaperones and mechanisms involved in prion infectivity these assays will miss a number of compounds that indirectly inhibit PrP conversion and aggregation (by binding to non-PrP targets). Additionally, compounds active in cell-free assays do not always exhibit cellular activity. However, these cell-free assays are useful for the high- throughput screening of large compound libraries and preliminary investigation of “mode of action”.

Fungal prion Models There are several prions in the yeast S. cerevisiae that have been found to be good models for research on the biology of prions.43, 71 The prion forms of Sup35p and Ure2p 13

give cells phenotypical differences to the normal Sup35p and Ure2p. These phenotypes are easily distinguished since the non-infected cells are red and the infected cells are white.22, 72 These assays are useful for targeting conserved mechanisms that are important for the propagation and infectivity of prions.72 The limitation of fungal models is that they may lead to the identification of active compounds specific for yeast prions. Guanidine hydrochloride (GuHCl) is an example whereby it has been shown to have a curing effect on yeast prions due to its inactivation of the Hsp104p prion chaperone in yeast. The targeting of Hsp104p, however, is not a viable option to inhibit prion propagation in mammals as Hsp104p is absent in mammals.73

The [URE3] and [PSI+] yeast strains have been adapted into a moderate-throughput screening assay that has allowed many compounds to be tested. Yeast assays are also much cheaper and more efficient pre-screening methods compared to mammalian cell- based assays.18, 74 Researchers at C.N.R.S. in France have screened a library of synthetic compounds for their effectiveness in curing yeast prion infections. From these results yeast prions have been shown to be a valuable model for an initial identification of new anti-prion compounds.43

While there has been little research done on the screening of compounds against yeast prions the work that has been done is showing promising results. Many of the compounds that have been identified with activity against yeast prions have also shown activity against mammalian prions. In addition, the activity from these fungal models seems to translate to in vivo mammalian activity much better than that of mammalian cell line assays.71 These fungal models are useful as a cheap and high-throughput first stage assays for the screening of large chemical libraries.

Animal Models A number of animal models have been developed using different mammalian prion strains. However, the use of these animal models in drug development is a slow and more expensive method with most of these assays requiring 50-300 days to complete.72 Due to this, these tests are usually only used as a final assessment. These models have mainly been developed in rodents instead of the animals that these prion diseases naturally occur in.75 Different scrapie strains are the most widely used screening strains; however more recently animal models have been developed to test against other prions including vCJD, CWD, and BSE. However, there are still some problems with these

assays, including ineffective transmission and the long incubation times required to measure the onset of symptoms.76

In rodent models inoculation is performed by the injection of homogenates prepared from clinically affected mice brains. Most studies have used either intracerebral or intraperitoneal inoculation. Using intracerebral inoculation the infection affects the central nervous system (CNS) immediately so the compounds are tested for their effectiveness in treating the prions in the CNS. Using intraperitoneal inoculation the infection first builds up in lymph and spleen tissue before infecting the CNS; compounds are then tested for their prophylactic protection.76, 77

A number of compounds have been investigated for their activity against prion-infected animals, however, to date there have been no compounds that have been effective if treatment is started at the first signs of symptoms.78 If treatment is started before or when the animal is inoculated some compounds have been shown to slow the progression of the disease.79-82 These animal models are useful for testing the ability of compounds to cross the blood-brain barrier (BBB) and inhibit the prion infection.83 In conclusion, while animal-based assay are slow and expensive they offer a good final screen for the effectiveness of compounds against mammalian prions.

ANTI-PRION THERAPEUTIC LEADS Most of the studies undertaken using the models described above have aimed to identify new drug leads that inhibit the infectious prion protein, reduce the proportion of toxic oligomers and amyloids in cells, and stabilise the protein in its native form.84, 85 The majority of this research has focused on screening FDA approved drug libraries so that compounds thus identified could be quickly developed into treatments. However, while a number of compounds with in vitro anti-prion activity have been identified none have proven to be effective for treatment in humans. The anti-prion compounds that have been identified can be classified into three different compound classes based on how they inhibit PrP. These classes are: 1. compounds that directly inhibit the conversion or aggregation of PrP, 2. compounds that effectively catalyse the clearance of the infectious PrPSc from the cells, and 3. compounds that inhibit cellular components or mechanisms important for prion propagation. Here, we review the literature on small anti-prion molecules that have, and are currently being, investigated.

1. Compounds that directly inhibit the conversion or aggregation of PrP The majority of the anti-prion compounds that have been identified inhibit the conversion of the PrPC into the infectious PrPSc. This can be through a variety of differing mechanisms including binding to PrPC or the PrPSc to inhibit their interaction or to stabilise the PrPC in its native form. Structural classes that have been identified include derivatives of sulfated polyanionic glycans (1-4) and azo dyes (5-10), tetrapyrroles (11-13), low molecular weight reagents (14-16), polyphenols (17-22), phenothiazine and quinoline derivatives (23-30), and a number of poly aromatic compounds that bind to PrP (31-38). These compounds have helped to greatly improve our knowledge of prions and prion mechanisms.

1.1 Sulfated polyanionic glycans The first anti-prion compounds were a number of sulfated polyanionic glycans (1-4).80 Numerous structure-activity relationship studies have been conducted using libraries of polyanionic glycans and a number of important molecular features of these compounds have been identified.86 These features include their molecular size and level of sulfation. As the size of the molecule and the level of sulfation increase the potency of the compounds increases.86

- OH OSO3 O R2 O R1 O O O O

-O3SO OH R3 1 3

- OSO3 OSO - O -O SO 3 n 3 NH O O HO O OH O O OSO - -O3SO 3 - O OSO3 2 4 OH Figure IV: Different polyanionic glycans that have been found to have anti-prion activity: pentosan- polysulfate (1), - dextran sulfate (2), the carrageenans (3), and heparin sulfate (4). NOTE: R1, R2 and R3 can be OH or OSO3 . Pentosan polysulfate (1) has been reported to be one of the most potent anti-prion glycans and the one with the most therapeutic potential. In Scrapie-infected mouse 86-88 neuroblastoma cells (ScMNB) pentosan polysulfate has EC50s under 1 nM. Additionally, in vivo studies have shown that this compound significantly increases the mean survival time of scrapie-infected mice.86, 89 A clinical trial of pentosan polysulfate (1), using cerebral catheter administration to by-pass the BBB, resulted in significant clinical improvement with no drug-related side effects.90 The patient additionally 16

survived 24 months longer than the median survival time after onset of symptoms. Even though a clinical improvement and an increase in survival was observed, brain atrophy continued to progress.90

Other active sulfated polyanions include dextran sulfates (2) with a molecular weight of 86 >500000 Da (ScMNB EC50 1 nM), the carrageenans (3), of which iota and lambda had 86 the greatest potency (ScMNB EC50 1-10 ng/ml) and heparin sulfate (4) and mimetics 86, 91 (ScMNB EC50 50-150 ng/ml). While these polyanionic glycans have potent in vitro activity they do not offer therapeutic potential due to a poor uptake by the infected organs.

Interestingly, these polyanionic glycans share common structural features with endogenous glycosaminoglycans (GAGs) in humans. GAGs contain long polysaccharide chains and are commonly located on the surface of cells where PrPC is located.92 GAGs are involved in a number of processes, including cell adhesion, cell growth, binding of cell surface proteins, viral entry and cancer metastasis.93 These GAGs have been linked to a number of types of amyloid deposits and there is strong evidence that these compounds are involved in the formation and stabilization of amyloids.94, 95 Due to the structural similarities between polyanionic glycans and GAGs it has been hypothesized that polyanionic glycans most likely bind to the PrPC and inhibit its interaction with endogenous GAGs that promote the deposition of amyloids.92

1.2 Sulfated azo dye derivatives A number of sulfated azo dyes and derivatives (5-10) are reported to be potent anti- prion compounds. Congo red (5) is a sulfated azo dye that binds to amyloid proteins and is used commercially for the staining of these amyloids. This compound is characterized by a planar, symmetrical shape with two sulfonate naphthalene groups connected by a biphenyl linker.96

Congo red (5) has potent in vitro activity, with an EC50 in the low nanomolar range 96-100 (ScMNB EC50 1-30 nM). However, congo red has not progressed as a therapeutic due to its in vivo degradation into toxic by-products as well as limited transport through the BBB.100 However, this prompted the therapeutic evaluation of several related analogues of congo red.101, 102

This has led to the discovery of the prion inhibitors Trypan Blue, Evans Blue, Sirius

Red F3B, Primuline, Thioflavin-S (6) and Chicago Sky Blue 6B (7) (ScMNB EC50 ~1 μM).103, 104 A number of other structurally-related compounds including curcumin (8, 17

105, 106 107-109 ScMNB EC50 10 nM), suramin (9, ScMNB EC50 12 μM), and dapsone (10) have also been reported to inhibit prions.110, 111 Many analogues have in vivo activity with little toxicity and have been shown to pass through the blood-brain barrier. However none of these compounds have shown potent in vivo activity after the onset of disease symptoms.97, 102 While these compounds have limited potential as treatment options, they serve as one of the most important tools for the study of amyloids.

- -O3S SO3 N N N N

NH2 H2N -OS + S N 5 + S OMe N N

- 6 -O3S N N SO3 N N MeO OH HO - -O3S NH2 H2N SO3 7 O O MeO OMe

O HO OH H H N N 8 N N H H O O O O O NH SO3H HO3S HN O S

H2N NH2 SO3H HO3S 10 SO3H SO3H 9 Figure V: Different azo dyes and related compounds that have been found to possess anti-prion activity: congo red (5), thioflavin-S (6), chicago sky blue (7), curcumin (8), suramin (9), and dapsone (10). Structural features of these molecules that are important for anti-prion activity are the planar orientation and the distance of the linking group from the two end moieties.97, 100 Additionally the anionic sulfonate or carboxylate on the naphthalene rings is an important structural feature.102 The importance of the anionic groups suggests that the mode of action of these compounds is similar to that of the polyanionic glycans.98 These azo dyes directly interact with PrPC to stabilise and block its interaction with GAGs. This inhibits amyloid formation by inhibiting seeding fractions from propagating.79, 99, 112

1.3 Cyclic tetrapyrroles A number of cyclic tetrapyrroles (11-13) have been reported to have potent anti-prion activity. These compounds are characterised by a framework of four interconnected pyrrole rings forming a cycle. These compounds are commonly found in nature and have huge biological importance, the most common being heme and chlorophyll. These compounds have previously been shown to bind strongly to proteins and affect their 3D conformation.82

A number of cyclic tetrapyrroles have potent anti-prion activity including duteroporphyrins (11, ScMNB EC50 1-10 μM), tetraphenyl porphines (12, ScMNB EC50 113, 114 0.5-10 μM) and phthalocyanines (13, ScMNB EC50 0.5-10 μM). These studies have also investigated derivatives of the compounds above with various metal complexes.113 The most potent cyclic tetrapyrrole is phthalocyanine tetrasulfonate which has an EC50 of 0.5 µM for scrapie-infected neuroblastoma cells.

R R R - -O3S SO3 R N HN N H N HN NH N N N H N NH N

R R -O S SO - R O R O 3 3 11 12 13

Figure VI: Different tetrapyrrole compounds that have been found to have anti-prion activity: duteroporphyrins (11), tetraphenyl porphines (12), and phthalocyanines (13). These tetrapyrroles share many structural similarities with azo dyes since they are hydrophobic, have a planar aromatic base scaffold and the inclusion of sulfonate groups leads to greater anti-prion activity. These structural features appear to be important for anti-prion bioactivity. Tetrapyrroles differ from azo dyes by being more soluble and having lower toxicity.

Whilst the tetrapyrroles potently inhibit PrPSc with low toxicity they are still not effective as therapeutics because they are ineffective in vivo at the later stages of prion diseases. There are many other cyclic and linear tetrapyrrole compounds that have not been screened against prions. These would offer a larger library for SARs and would help in the discovery of new therapeutic leads. In conclusion, these compounds offer a good starting point for the design and development of new anti-prion therapeutics.

1.4 Chaperone-like chemical reagents (<100 Da) A number of chaperone-like reagents (14-16) have been reported to decrease the rate of conversion of PrPC into PrPSc. Dimethyl sulfoxide (DMSO, 14), a low molecular weight chemical reagent, promotes the clearance of amyloids involved in a number of different neurodegenerative diseases.115 In clinical trials of DMSO on patients with amyloidosis it proved to have a statistically significantly effect on amyloid accumulation.116

- O OH O + S HO OH N

14 15 16 Figure VII: Different low weight chaperone-like reagents that have been found to exhibit anti-prion activity: DMSO (14), glycerol (15), and TMAO (16). The chemical reagents that are reported to successfully inhibit conversion of PrPC to PrPSc include DMSO, glycerol (15) and trimethylamine N-oxide (TMAO, 16). These compounds moderately inhibit PrPSc when added to the culture media of scrapie- infected mouse neuroblastoma cells. It is thought that these chemical reagents stabilise the soluble PrPC thereby inhibiting its conversion to PrPSc.117

DMSO (16) was tested on prion-infected hamsters in vivo and was shown to successfully delay the onset of disease symptoms. This suggests the use of DMSO in a multi-drug therapeutic approach. However, toxic effects of DMSO were observed in hamsters upon long-term treatment with high doses (>55 days).118 It is interesting to note that while the anti-prion activity of DMSO has been identified in mammalian models, the same activity has not been reported in fungal models.

1.5 Polyphenolic derivatives A number of natural polyphenolic compounds (17-22) are potent inhibitors of PrPSc. These compounds are commonly produced in plants and bind to and precipitate protein- complexes.119 These commonly found polyphenols can inhibit PrPSc propagation in cell cultures at nanomolar doses.120

The most active polyphenol is tannic acid (17, ScMNB EC50 100 nM), other active polyphenolic compounds include the flavonoid-like compounds katacine (18, ScMNB

EC50 500 nM), epicatechin monogallate (19, ScMNB EC50 1-10 μM) and 120-122 epigallocatechin 3,5-digallate (20, ScMNB EC50 1-10 μM). Similar to previous compounds, the activity of polyphenols increase as the molecular size and number of phenolic rings increases. Indeed, a number of smaller polyphenolics such as epicatechin and epigallocatechin, exhibit no anti-prion activity.120 A number of the higher molecular weight polyphenols have been screened in animal models, but show no significant 20

therapeutic effect.121 This is most likely due to the inability of these compounds to cross the BBB.

Initially, the smallest polyphenol with anti-prion activity was identified as 2,3,5,7,3',4'- 120 pentahydroxyflavan (21, ScMNB EC50 1-10 μM). However, recent studies have screened polyphenolic extracts of different plants against prions in cell culture and animal models. The even lower molecular weight flavonoid, baicalein (22, ScMNB

EC50 ~40 μM) which has fewer hydroxyl groups than pentahydroxyflavan, was identified as an active anti-prion compound in vivo.123 OH HO OH HO OHO OH HO O OH O O OH O O OH O O HO O OH HO O OH OH O O O OH HO O O OH O O O HO OH HO O OH OH O OH OH HO OH HO OH O HO OH OHO OH HO O OH OH O OH 18 HO OH OH OH HO OH OH OH HO O 17 OH OH HO O OH OH HO O OH OH O 21 O O OH O O HO O OH OH O OH OH OH HO OH OH O OH OH 19 20 22 Figure VIII: Some of the polyphenolic compounds that have been found to have anti-prion activity: tannic acid (17), katacine (18), epicatechin monogallate (19), epigallocatechin 3,5-digallate (20), 2,3,5,7,3’,4’-pentahydroxyflavan (21), and baicalein (22).

While polyphenolic compounds exhibit nanomolar activity, these compounds are not viable therapeutic options. The larger, more potent polyphenols are not active in vivo and none of the other polyphenols are effective after onset of disease symptoms.121 Additionally, these polyphenols are likely to exhibit non-specific protein binding and to be quickly metabolised in animals. However, smaller polyphenolic flavonoids could be a good starting point for the development of anti-prion therapeutics.

1.6 Phenothiazine and quinoline derivatives Phenothiazine, acridine and quinoline derivatives (23-30) are the most extensively studied anti-prion compounds.109 Since their discovery, these compounds have generated a great deal of interest, primarily due to these compounds being FDA- approved drugs, of which many efficiently cross the blood-brain barrier with low toxicity. Additionally, a number of these derivatives are already used for treatment of neurological disorders.124

Quinacrine (23) and chloroquine (24) were the first identified to have anti-prion 124 125 activity with EC50 values of 0.3 μM and 2.3 μM, respectively. Interestingly, these compounds only have weak activity in yeast-based anti-prion assays.43, 126 A large number of phenothiazine derivatives including chlorpromazine (25), promethazine, 120, 126, 127 promazine and acepromazine have potent activity (ScMNB EC50 <1-10 μM). Quinine (26) and quinoline derivatives, many of which are current treatments for 128 malaria, also show potent activity (ScMNB EC50 <1-10 μM). Similar compounds phenanthridine (27) and tacrine (28) were identified using fungal-prion screening.43, 129 Interestingly, the larger molecular size bisquinolines (29) and bis-acridine (30) are more 130-132 active in vitro (ScMNB EC50 <1-25 nM).

N N HN HN S O Cl N

Cl N Cl N N 23 24 25

HO Cl N NH2 O

N N NH2 N 26 27 28 Cl Cl Cl OMe

N H N N O N H N O N H N N N H H 29 OMe 30 Cl Figure IX: Some of the most active phenothiazine, acridine and quinoline derivatives that have been found to have anti-prion activity: quinacrine (23), chloroquine (24), chlorpromazine (25), quinine (26), 6-amino-8-chloro- phenanthridine (27), tacrine (28), bisquinoline (29) and bisacridine (30).

While these compounds have potent in vitro activity they show no therapeutic effect in animal models83, 133 and failed to slow disease progression and onset of disease symptoms in clinical trials.134, 135 More recently, in an animal model, quinacrine, administered using a cerebral catheter to bypass the BBB, delayed onset of symptoms when used in high (toxic) concentrations.83

More recent studies have focused on understanding the mode of action of these compounds. However, these studies have resulted in conflicting conclusions as to whether or not the inhibition of the PrPSc is through direct binding.109, 126 Early cell-free conversion assays indicated that these compounds do not bind directly to PrPC to inhibit conversion.71 However, later studies reported the binding of acridines and quinolines to PrPC.124 While the mode of action of these compounds has not yet been properly defined; all studies have concluded that these compounds somehow inhibit the conversion of PrPC into PrPSc.120 Currently, however, these compounds do not offer therapeutic potential due to their limited effectiveness in vivo.

1.7 Poly-aromatic PrPC binding compounds Screening efforts have also led to the identification of a number of other aromatic PrPC binding compounds. These include derivatives of benzylidene-benzohydrazide (31), diphenyl-pyrazoles (32), guanabenz (33), and indole-glyoxylamides (34).

OH N NH H N N OH O O O Br 31 32

Cl O H O N NH2 N N N N NH H Cl N 33 H 34

Figure X: Different aromatic PrP-binding compounds that have been found to have anti-prion activity: benzylidene- benzohydrazide 293GO2 (31), diphenyl-pyrazole anle138b (32), guanabenz (33), and indole-glyoxylamide (34). Benzylidene-benzohydrazide derivatives were identified by screening 10,000 compounds in a cell-free aggregation inhibition assay. These compounds were identified to bind to PrPC in vitro and exhibit anti-prion activity in cell cultures. Structure-activity relationships led to the discovery of the most active compound 136-138 293GO2 (31, ScMNB EC50 ~2 μM). It was found that a naphthalen-2-ylmethylene on one side and hydroxyl groups in the meta or para positions of the phenyl rings led to 23

an increase in potency. Later, diphenyl-pyrazoles were identified by screening a further 10,000 compounds.136 Further screening and analysis of structure-activity relationships led to the identification of the lead compound, anle138b (32, vCJD-infected human 139 neuroblastoma cells EC50 7.1 μM). Further studies on anle138b reported a significant increase in the survival time of mice infected with the human vCJD. To date, anle138b has shown the best in vivo anti-prion selective activity, however it is still early in its therapeutic development.139

The benzylidene-benzohydrazide and the diphenyl-pyrazoles were found to have their effect through the binding to PrPC and inhibition of PrPC oligomerisation.139 Additionally, these compounds have potent activity against polyglutamine protein aggregation in zebrafish, α-synuclein aggregation, and β-amyloid aggregation, also through the inhibition of protein oligomerisation.137, 139-142

Guanabenz (33) was identified as a potent anti-prion compound in a large screen for compounds with activity against yeast prions.129 This study also examined the activity of guanabenz against mammalian prions, identifying high potency in vitro (ScMNB

EC50 <10 μM) and in vivo, with guanabenz displaying the best increase in survival time of any compound.129 However, guanabenz has not progressed further due to a high level of cellular toxicity. More recent studies have focused on the identification of less toxic derivatives of guanabenz that may be useful for therapeutic development.143, 144

Indole-glyoxylamide (34) derivatives were identified in a screen that aimed to specifically identify drug-like therapeutics for prion diseases.145 The indole- glyoxylamide scaffold was used due to the relatively high number of compounds containing the scaffold that have progressed to clinical trials.146 Structure-activity relationship analysis has been performed through the synthesis of over 100 derivatives 145, 147 and this led to a high level of potency (ScMNB EC50 ~1-10 μM). These recently identified compounds have not yet been studied further and their bioactivity in vivo is undetermined.

1.8 “Virtually” identified poly-aromatic PrPC binding compounds Since the three dimensional structure of PrPC was deduced late in the 1990s and PrPSc not long after, there has been an increase in the number of compounds that have been designed in silico through virtual screening of molecular fragments for the ability to bind to the PrP.67, 68, 148, 149 This process was first used in 2000 with a library of 210,000

compounds “virtually” screened to identify PrPC binders. The virtual hits were then screened using cell-free binding assays, cell line assays and cell viability assays. This led to the identification of a dicarbonitrile compound (cp-60, 35) and a methoxybenzene sulfonohydrazide compound (cp-62, 36).150 Various dicarbonitrile derivatives exhibit high potency with many being put forward as possible drug candidates (ScMNB EC50 5- 20 μM).69, 150-152

NH 2 N N Cl N O N S O H S N Cl NH O N O 2 O H N O 35 36

S HO NH O O N N N N N N HO H H 37 38 Figure XI: Different PrPC binding compounds that have been found to have anti-prion activity discovered using virtual screens: dicarbonitrile Cp60 (35), methoxybenzene sulfonohydrazide Cp62 (36), GN8 (37), and diaryl-2- aminthiozole (38). Since this initial screen, virtual screening has become a routine method for the development of PrPC binding compounds. In addition, this virtual screening approach aids in the design and synthesis of derivatives that typically have a similar three- dimensional placement of molecular binding groups with differing core structures.153-156 This approach has led to the identification of 2-pyrrolidin-1-yl-N-[4-[4-(2-pyrrolidin-1- yl-acetylamino)-benzyl]-phenyl]-acetamide (GN8) derivatives (37) and diaryl-2- aminothiazole derivatives (38).136, 157, 158 These compounds have been extensively 159 studied with the most potent of the GN8 derivatives having EC50s from 0.5 - 1 μM and the diaryl-2-aminthiozole having bioactivity in the nanomolar range (EC50s from 0.1 – 0.5 μM).158, 160, 161 Diaryl-2-aminothiazole has been evaluated further using animal models and has exhibited a significant increase in survival times.160, 162

These compounds are highly effective and are typically highly selective as they inhibit conversion of PrPC to PrPSc through direct binding. While these binding compounds are highly potent in vitro, a large amount of development is needed to obtain lead compounds that are active in vivo.155 This virtual screening approach also excludes a number of other potential targets for PrPC inhibition and this limits the chemical diversity of the prion inhibitors that can be identified in this way.

2. Compounds that enhance the clearance of PrPSc from cells Very few studies have been conducted on compounds that enhance the clearance of PrPSc from cells. This includes compounds that catalyse the transport of PrPSc out of cells or compounds that render the PrPSc protease-sensitive so that it can be destroyed by the cells proteolytic machinery. To date, compounds that significantly clear the infectious PrPSc from cells include a number of polyamine compounds (39-42) and tetracyclines (43-45).

H2N NH2

NH HN O O

NH2 N

HN O O NH

H O H O N N N N NH2 H2N N N N N O H O H

HN O O HN N NH2 +H3N O O

NH HN + +H3N N NH3

H2N NH2 + 39 N N NH3 +H3N N N

+ NHEt2 +H3N N NH3 Et2HN HN HN NH + P S 3 N NHEt2 40 N NHEt Et2HN 2 S HNHN HN P N P NHN N N S Et2HN P N N P P N S N P N N H N NHEt2 Et2HN N NH N H P NH N S Et2HN N S P NH NHEt2 NH NHEt2

Et2HN 41 Figure XII: Different dendrimers that have been found to have anti-prion activity: polyamidoamide 1.0 (39), polypropyleneimine (40), and phosphorus containing polyamine dendrimer 1.0 (41).

2.1 Polyamine compounds A number of polyamine dendrimers have been shown to inhibit PrPSc propagation. These dendrimers are characterised as having repeating polymers that branch symmetrically from a base. This generally gives dendrimers a spherical three- dimensional shape. Dendrimers can be classified according to the number of generations of branch points from the core.163 Active polyamine dendrimers include polyamidoamide (PAMAM, 39), polypropyleneimine (PPI, 40) and polyethyleneimine (PEI).164 Later, phosphorous-based polyamine dendrimers (41) were also found to be active against prions.165 The larger fourth-generation of these dendrimers are the most potent with EC50s against ScMNB under 50 nM, while the first-generation dendrimers 165, 166 are typically ~1000-fold less potent (EC50s >10 μM).

Structure-activity relationship analysis has indicated that an increase in the generation of these dendrimers results in an increase in the anti-prion activity. This is likely due to the increase in the number of reactive groups on the surface of the dendrimers. This theory was validated by a loss of activity when the cationic amine groups were replaced with less reactive hydroxyl groups.167, 168

While these compounds can successfully inhibit PrPSc propagation at low doses with no toxic effects, these compounds cannot cross the BBB because of their size and physicochemical properties.168, 169 Recently, however, modified PPI dendrimers (37) with reactive maltose groups have been shown to cross the BBB.170 This indicates that dendrimers represent a good molecular scaffold for the development of anti-prion therapeutics. These dendrimers are hypothesised to directly bind to the pathogenic PrPSc 167, 168 thereby rendering it protease-sensitive and allowing secondary lysosomes to proteolytically degrade the PrPSc.163

N O

N N N H H CH2SO3H N N

N 42 Figure XIII: The anti-prion tyrosine kinase inhibitor imatinib mesylate (42). The polyamine tyrosine kinase inhibitor imatinib mesylate (42) has also been found to efficiently enhance the clearance of PrPSc from cells. This compound is highly potent, with EC50s against ScMNB of less than 1 μM. It is thought to activate lysosomal

degradation of the PrPSc to significantly decrease its half-life in these cells.171 Subsequently, imatinib mesylate was evaluated in a scrapie-infected mouse model. Imatinib mesylate treatment was found to result in a significant increase in the survival time of the mice.172 Since these studies little work has been reported on analogues of this compound or other tyrosine kinase inhibitors. However, it has been noted that the size and pharmacokinetic properties of imatinib mesylate make it a suitable candidate for drug development.171

2.2 Tetracyclines Tetracyclines were first observed to inhibit amyloid formation during human trials of anthracycline 4'-iodo-4'-deoxy-doxorubicin (IDX, 43) on cancer patients.173 Later other tetracyclines were shown to be highly active against mammalian prions with low toxicity.81, 174 Anti-prion tetracyclic compounds include: tetracycline (44),65 doxycycline 175-177 (45), and minocycline (ScMNB EC50 ~100 μM). Further studies identified these compounds as highly active in vivo.174 These tetracycline compounds have low toxicity and are able to cross the BBB,178 thus doxycycline was evaluated in clinical trials for spontaneous CJD (sCJD). In the first clinical trial doxycycline treatment resulted in an increase in patient survival time of approximately 7 months.179 A second, larger, randomised, and double-blinded trial of doxycycline versus a placebo provided contradictory results with no significant increase in patient survival time observed.180 This was significant as it was the largest clinical trial of an anti-prion drug lead to have been conducted.

O OH O OH OH OH O OH O O OH OH O O OH OH OH

NH2 NH2 O O OH O O OH O H H H H OH N OH N NH I 2 44 43 45 Figure XIV: Different tetracycline compounds that have been found to have anti-prion activity: anthracycline 4’- iodo-4’-deoxy-doxorubicin (43), tetracycline (44), and doxycycline (45). While the mode of action of tetracyclines is not well understood, it is thought that these compounds have a direct effect on the PrPSc. When purified PrPSc aggregates are incubated with tetracyclines the protease-resistance of the PrPSc is significantly decreased. This suggests that these compounds promote the breakdown of the PrPSc aggregates.176

3. Compounds that target cellular components important for PrP A number of anti-prion compounds have been identified that have their activity through the inhibition of cellular components that have important functions in the switching, aggregation and/or propagation of PrPSc. Most of these anti-prion compounds have their effect through the reduction of cholesterol levels or the reduction of redox-active metal levels in cells.

3.1 Cholesterol Inhibitors A number of cholesterol lowering drugs have been discovered to have anti-prion activity. These compounds include a number of polyene drugs (46-48), statin drugs (49- 50), squalestatin (51) and verapamil (52). It is theorised that the depletion of cholesterol indirectly increases the autophagocytic removal of PrPSc. 181-183 The depletion of cholesterol effectively alters the protein : lipid ratio in the membrane microdomains where prion switching occurs. It is thought that this in turn alters lysosomal and endosomal membrane trafficking in the cell leading to a decrease in PrPSc levels.184

The first cholesterol lowering compound to be shown to have anti-prion activity was the polyene antimycotic amphotericin B (AmB, 46). This compound was discovered to have anti-prion activity when a small library of 35 drugs including various antivirals, antibiotics, anti-parasitics and anti-fungals were screened against prions.185, 186 AmB lowers cholesterol levels by direct binding to sterols, thereby altering the sterol composition of the cells.184

AmB (46) has potent activity in vitro at nanomolar concentrations and187 is one of the few compounds that has been shown to significantly increase survival time in mice when used to treat scrapie at late stages after onset of disease symptoms. Like other compounds, while this activity is significant, it does not stop the progression of the disease.187, 188 In mouse scrapie models, AmB has a significant effect when used to treat intra-cerebral inoculated mice.187, 189 This indicates that AmB can successfully penetrate the blood-brain barrier in sufficient quantity. A similar polyene compound that has been 190 shown to have anti-prion activity is filipin (47, ScMNB EC50 2 μM). Treatment with AmB, however, is still not a successful therapy due to acute toxicity.191 MS-8209 (48) is an analogue of AmB that has similar antifungal activity with lower toxicity and greater water solubility. MS-8209 was shown to be slightly more potent than AmB in animal models of prion disease and has therefore been selected as a new therapeutic lead.191, 192

OH OH HO O

O O OH OH OH OH O OH

O O 46 HO OH OH NH2 OH O OH

HO O OH OH OH OH O O

O O

48 HO OH HN

OH OH OH OH OH OH OH O H OH O OH O O O HO OH 47

Figure XV: Different polyene compounds that have been found to have anti-prion activity: amphotericin B (46), filipin (47), and MS-8209 (48). The statins were identified as another group of cholesterol lowering drugs with anti- prion activity not long after AmB.193 Statins include a number of drugs that are currently used for the inhibition of cholesterol biosynthesis and lowering of blood cholesterol levels. They reduce cholesterol biosynthesis through the inhibition of 3- hydroxy-3-methyl-glutaryl-coenzyme A reductase.194 Lovastatin was identified as 193 having potent in vitro anti-prion activity (49, ScMNB EC50 500 nM). Further studies confirmed that the anti-prion activity of lovastatin was due to the inhibition of cholesterol biosynthesis. Lovastatin had no activity in cell-free assays and lost its anti- prion activity when cholesterol levels were increased artificially.120 Further in vivo studies with simvastatin (50) resulted in a significant increase in the survival time of PrPSc infected mice and slower progression of loss of motor funtion.195, 196 It was also later reported that treatment with lovastatin significantly decreases amyloid formation in vitro and could be used in combination therapy for multiple neurodegenerative disorders.197

Other compounds that have been found to have anti-prion activity due to their ability to lower cholesterol levels include: squalestatin (51) and verapamil (52). Squalestatin inhibits cholesterol biosynthesis by inhibiting qualene synthase. This compound was 30

reported to have potent anti-prion activity in vitro and in vivo and to significantly increase the survival time of scrapie-infected mice.198, 199 Verapamil is a calcium- channel blocker that blocks the recycling of cholesterol from the plasma membrane to the endoplasmic reticulum. Verapamil was shown to have potent anti-prion activity in 200 vitro (ScMNB EC50 15 μM). Later, it was reported that verapamil also exhibits potent anti-prion activity against yeast prions, suggesting that the sterol-rich membrane microdomains required for prion switching in mammalian cells may be conserved in yeast and play a role in yeast prion switching.43 Squalestatin and verapamil may have therapeutic potential, however they have not yet been studied further.

O HO O HO O O O O O O

O O H H HO O O O CO2H

CO2H HO2C OH 49 50 51

CN N OMe

MeO OMe HCl OMe 52

Figure XVI: Other cholesterol lowering drugs that have anti-prion activity: lovastatin (49), simvastatin (50), squalastatin (51) and verapamil (52).

3.2 Chelators of redox-active metal The cellular prion protein in humans is a copper-binding glycoprotein that is highly expressed in neuronal cells.201, 202 The binding of certain transition metals, in particular copper (II), to PrPC has been hypothesised to influence the process of prion conversion in cells. This copper-binding glycoprotein and similar redox-active-metal-binding proteins have also been associated with diseases such as Parkinson’s disease and Alzheimer’s disease.201 Other redox-active metals that have been reported to be important for the pathogenesis of PrP and associated diseases include Zn2+ and Fe3+.203 In 2003, Sigurdsson et al. reported that by reducing the levels of Cu2+ in scrapie- infected mice through treatment with the copper chelator d-penicillamine (53) the onset of the disease was significantly delayed. This study therefore confirmed that Cu2+ ions influence prion disease mechanisms. More broadly it supported the view that transition

metal ions (primarily Cu2+, Zn2+ and Fe3+) may promote important prion disease mechanisms.204

Since this study, a number of different metal-chelating compounds have been shown to have anti-prion activity. D-penicillamine (53), clioquinol (54), cimetidine (55), neocuproine (56), bathocuproine (57), 2,2’-biquinoline (58) and a number of the porphyrins described above are all copper-chelating compounds that have been shown to have anti-prion activity.205-207 These compounds bind to copper with a 1:1 molar ratio 207 and have EC50s in the nanomolar range (as determined using ScMNB cells). These compounds were also evaluated for their ability to inhibit the activity of copper- dependent superoxide dismutase (SOD). SOD is another protein whose misfolding is associated with neurodegenerative diseases. It was found that those compounds that had higher anti-prion activity also had higher SOD-inhibition activity.207

Cl O HN HO SH N N I N S NH2 N N N OH H H 53 54 55

N N N N N N 56 57 58

Figure XVII: Metal-chelating compounds that have anti-prion activity: D-penicillamine (53), clioquinol (54), cimetidine (55), neocuproine (56), bathocuproine (57) and 2,2’-biquinoline (58). While metal chelators are extremely effective in inhibiting the formation of PrPSc amyloids in vitro they are not effective as anti-prion therapeutics. This is due to the toxic side-effects associated with depletion of copper to physiologically deficient levels, not only in the brain but also in other organs such as the liver. For these compounds to be effective as therapeutics, the depletion of these redox-active metals needs to be targeted more specifically to the brain.51

Conclusions It can be seen that over the last 30 years there has been a large focus on the screening of compounds to find new therapeutic leads for the treatment of diseases caused by prions. While many anti-prion compounds have been found, they represent a relatively low level of chemical diversity. One reason for this could be that libraries containing small synthetic compounds with similar structural motifs have been screened. A large number 32

of the compounds that directly inhibit the conversion and/or aggregation of the prion protein have many structural similarities. The majority of these compounds are based on a simple poly-aromatic ring scaffold. This may be due to hydrophobic interactions with the protein that are important for binding. The addition of anionic sulfonate groups also seems to correlate with an increase in anti-prion potency. Several compounds that antagonise prion propagation in vitro or in vivo have limited therapeutic suitability, primarily due to their poor passage through the blood/brain barrier and/or severe toxicity. Of the few compounds that have been found to efficiently cross the blood-brain barrier and that have low toxicity none have been found to be effective in vivo after the onset of disease symptoms. Therefore, there is a need for new compounds to develop into therapeutics for prion diseases. Recently, it has been proposed that large scale screening studies to date have failed to identify compounds with successful in vivo activity due to the use of poorly designed mammalian cell-line models.58 The development of novel anti-prion assays for the screening of novel compound libraries or extracts derived from plants, animals and microbes could therefore be a lucrative source of anti-prion leads with greater chemical diversity.

NATURAL PRODUCT DRUG DISCOVERY A natural product can be defined as a secondary metabolite synthesized by a living organism that is not necessary for primary metabolic functions. These metabolites typically enhance the survival of an organism through chemical defence, competition and communication.208 Natural products have been used commercially for many applications throughout history. The most significant application has been the use of natural products as therapeutics to treat various diseases. Approximately 51% of marketed drugs over the last 30 years are or have been inspired by natural products. Drugs that are ‘naturally inspired’ include natural product derivatives and synthetic products that mimic natural products.209 Some examples of historically important drugs that have been developed from natural sources include the pain killer morphine (55, isolated from the opium in Papaver somniferum poppy),210 the antibiotic penicillin (56, isolated from the mould Penicillium notatum), the anticancer compound taxol (57, isolated from the pacific yew tree Taxus brevifolia)211 and the antimalarial artemisinin (59, isolated from the plant Artemisia annua).212, 213 Major focuses of natural product

drug discovery studies have been to find compounds that can be used as treatments for bacterial and fungal infections, cancers, parasitic disease and viral infections.209

(a)

(b) HO O H N H S O H N O N OH HO O

59 60

O O HO O H O H H O O NH O O O O H H O O OHO O O O 61 62

Figure XVIII: (a) A pie chart showing the proportion of all new drugs over the last 30 years that have been developed from various sources. This figure is adapted from Newman and Craig, (2016).209 (b) Common drugs that have been developed from natural products: morphine (59), penicillin G (60), taxol (61) and artemisinin (62).

Anti-prion natural products While natural products have undoubtedly been a huge source of bioactive drug-like molecules, no studies to date have screened libraries of natural product extracts for the isolation of novel anti-prion compounds. This is surprising when one considers that a large number of the anti-prion therapeutic leads discussed earlier are, in fact, natural products or natural product derivatives. In our systematic review of reported anti-prion compounds we found that 44% of the anti-prion lead therapeutic scaffolds are natural products or natural product-related synthetic products. This is high considering that 34

most studies have focused on the screening of drug and combinatorial libraries rather than natural product libraries.

Figure XIX: A pie chart displaying the origin of the anti-prion compounds that have been identified. Additionally, in our review of known anti-prion compounds we point out that the known anti-prion compounds display a low chemical diversity. Many of these compounds have similar features, such as poly-aromatic or cyclic core scaffolds with substitution of a limited range of anionic moieties. Natural product libraries represent a much larger chemical diversity than that of synthetic combinatorial libraries. This high chemical diversity is due to the highly complex and three-dimentional shaped molecules that are synthesized by living organisms for specific biological roles.208 Feher et al. (2003), compared the structural properties of three different classes of compounds: synthetic combinatorial products, natural products and currently used drugs.214 Their analysis indicated that not only did natural products occupy a more diverse chemical space, but they also had a closer match to currently used drugs (Fig. XX).214 With current therapeutic leads having a low chemical diversity, screening of natural extracts with a high chemical diversity is essential for the identification of novel therapeutic entities.

Figure XX: Principal component analysis of structural features of the compound libraries. This figure is reproduced from Feher et al. (2003). a) the diversity of synthetic combinatorial compounds, b) the diversity of natural products, and c) the diversity of drugs in current clinical use.215

It is now becoming clear that prions are common in nature and play important biological roles.9, 32 Therefore, one could hypothesize that diverse organisms produce compounds for the regulation and formation of these prions. The screening of crude natural extract libraries has been significantly hindered by the limitations of the assays available for screening such complex mixtures. However, the development of new high throughput anti-prion assays can facilitate the screening of this largely untapped chemical resource.

CONCLUSION Prion diseases are a rare group of fatal neurodegenerative diseases caused by infectious misfolded proteins. Currently, there are no treatment options to stop or slow the progression of these diseases. Effective therapy for these diseases has been significantly hindered by the lack of knowledge about prions and the late diagnosis of these diseases. Recent research suggests the existence of strong correlations between prion-like mechanisms and a number of other more common neurodegenerative diseases, such as Alzheimer’s disease, Parkinson’s disease and Motor Neurone disease. Therefore, it is of great interest to identify anti-prion compounds that may be useful for the treatment of a number of neurodegenerative disorders.

The current lead compounds that have been identified and evaluated for their use as therapeutics for prion diseases have failed to be successful in vivo when administered after the onset of disease symptoms. A number of factors have contributed to this lack of curative therapy, including the lack of suitable assays for screening compound libraries to identify successful lead candidates. A number of the currently identified lead compounds are unable to pass the BBB and/or have neurotoxic side effects. Unfortunately, due to the predominant use of synthetic combinatorial and drug libraries in anti-prion screening studies, there is a lack of chemical diversity in the lead anti-prion compounds that have been identified to pass the BBB with a low toxicity. A high percentage of the compounds that have been identified in previous anti-prion screens are related to natural products. Therefore, we suggest that screening natural product extract libraries could be a fruitful approach for the discovery of novel anti-prion molecules.

AIMS, OBJECTIVES AND SCOPE OF THE STUDY The literature review reveals a number of major gaps in anti-prion research. As such there are three major aims in this project.

The first aim of this PhD project is develop a new screening method to identify anti- prion compounds and to then screen a natural product extract library for anti-prion activity. There are several criteria that need to be fulfilled to develop an effective screen. These include a high-throughput microtiter plate layout so that the screen can be done using robotic methods. In addition, the screen needs to suitable for screening in PC2 laboratories with a low cost per assay. The last requirement is that the assay be optimised for complex mixtures of chemicals that are commonly found in natural extracts. By developing a high throughput anti-prion assay and screening a library of natural extracts we aim to prove our hypothesis that the natural environment is potentially a fruitful source of novel anti-prion compounds.

The second aim of this project is to isolate and identify compounds in any active extracts that are responsible for the observed anti-prion activity. Using chromatography and spectroscopic methods we aim to purify and identify a number of different unique anti-prion natural products. We hope to find novel anti-prion structures that will increase the chemical diversity of know active compounds.

The third and final aim is to identify if any of the compounds would make satisfactory lead compounds for further development. With current anti-prion compounds primarily failing due to poor blood-brain permeation and toxicity, this will include further studies to evaluate if the compounds identified have better potential as anti-prion therapeutic leads.

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CHAPTER 1:

Yeast-Based Screening of Natural Product Extracts Results in the Identification of Prion Inhibitors from a Marine Sponge

STATEMENT OF CONTRIBUTION:

This section includes a co-authored research paper, ‘Yeast-Based Screening of Natural Product Extracts Results in the Identification of Prion Inhibitors from a Marine Sponge.’ that has been accepted and publish in Prion. The reference of the co-authored manuscript is:

Jennings, L. K.; Ahmed I.; Munn A. L.; Carroll A. R.; “Yeast-Based Screening of Natural Product Extracts Results in the Identification of Prion Inhibitors from a Marine Sponge”. Prion 2018, 234-244.

My contribution to the paper involved the physical experimentation and organization of information, data and references, as well as the preparation of the manuscript.

(Signed)______

Laurence Kane Jennings Date:______

(Signed)______

Corresponding author: Anthony R. Carroll Date:______

(Signed)______

Supervisor: Anthony R. Carroll Date:______

PRION PUBLICATION:

CHAPTER 2:

Anti-prion Butenolides and Diphenylpropanones from the Australian Ascidian Polycarpa procera

STATEMENT OF CONTRIBUTION: This section includes a co-authored research manuscript that has been prepared for submission to Journal of Natural Products; “Anti-prion Butenolides and Diphenylpropanones from the Australian Ascidian Polycarpa procera.” The details of the co-authored manuscript are:

Jennings, L. K.; Robertson, L. P.; Rudolph K. E.; Munn A. L.; Carroll A. R.; Anti-prion Butenolides and Diphenylpropanones from the Australian Ascidian Polycarpa procera. J. Nat. Prod. In preparation for submission

My contribution to the paper involved the physical experimentation and organization of information, data and references, as well as the preparation of the manuscript.

(Signed)______

Laurence Kane Jennings Date:______

(Signed)______

Corresponding author: Anthony R. Carroll Date:______

(Signed)______

Supervisor: Anthony R. Carroll Date:______

JOURNAL OF NATURAL PRODUCTS MANUSCRIPT:

Anti-prion Butenolides and Diphenylpropanones from the Australian Ascidian Polycarpa procera

Laurence K. Jennings,†,‡ Luke P. Robertson,†,‡ Kathryn E. Rudolph,‡ Alan L. Munn,┴ Anthony

R. Carroll*,†,‡,§

†Environmental Futures Research Institute, Griffith University (Gold Coast campus),

Parklands Drive, Southport, QLD 4222, AUSTRALIA.

‡School of Environment and Science, Griffith University (Gold Coast campus), Parklands

Drive, Southport, QLD 4222, AUSTRALIA.

§Griffith Institute for Drug Discovery, Griffith University (Brisbane Innovation Park), Don

Young Road, Nathan, QLD 4111, AUSTRALIA.

┴Menzies Health Institute Queensland, Griffith University (Gold Coast campus), Parklands

Drive, Southport, QLD 4222, AUSTRALIA.

*[email protected]

ABSTRACT: A library of 500 Australian marine invertebrate extracts were screened for anti-prion activity using a yeast-based assay and this resulted in an extract from the ascidian

Polycarpa procera showing potent activity. Purification of this extract led to the isolation of six new butenolide metabolites, the procerolides (1-4) and two related diphenylpropanones, the procerones (5-6) as the bioactive components. The structures of 1-6 were elucidated from the analysis of 1D/2D NMR and MS data and their absolute configurations determined from comparison of experimental and computed ECD data. Compounds 1-6 were tested for anti- prion activity in a yeast-based assay and 1 and 5 displayed potent bioactivity (EC50 of 22.7 and 29.4 μM, respectively) comparable to the potently active anti-prion compound, guanabenz. The procerolides and procerones are the first anti-prion compounds to be reported from ascidians indicating that ascidians may be an untapped source of new lead anti-prion compounds.

O OH

O Br

OH Br MeO Br Procerolide A (1)

O HO Br

Polycarpa procera OH Br MeO Br Procerone A (5)

Prion diseases are fatal, neurodegenerative disorders caused by misfolded proteins. The human prion protein forms amyloid plaques which build up in the brain and cause cell damage leading to neurodegeneration.1 In the United Kingdom in the 1980s an outbreak of the prion disease, variant Creutzfeldt-Jakob disease (vCJD) occurred. Patients contracted the disease from eating tainted meat obtained from cattle suffering from bovine spongiform encephalopathy (BSE). With no treatment options for this disease the outbreak prompted an extensive search for anti-prion therapeutics. However, to date, no effective treatment has been developed for this fatal disease.2

Over the last 60 years the marine environment has been a prolific source of bioactive compounds.3 Marine ascidians in particular have been shown to contain unique chemical diversity and some ascidian derived compounds have been developed as promising leads for the design and development of new drugs.4 However, neither ascidian extracts nor their natural products, have been evaluated for anti-prion activity. We have developed a microplate high throughput yeast-based anti-prion assay and used it to screen a library of 500 Australian sub-tropical marine macro-organism extracts. This led to the identification of an anti-prion active extract from the ascidian, Polycarpa procera.5 Herein, we report the screening, isolation, structure identification and anti-prion bioactivity of a series of brominated butenolides and diphenylpropanones from P. procera named the proceolides and procerones, respectively.

RESULTS AND DISCUSSION

A freeze-dried sample of P. procera was exhaustively extracted with MeOH. The LC-MS trace of the extract displayed a number of peaks possessing brominated ion clusters. The extract was separated by repeated semi-preparative HPLC on C18 silica gel eluting with varying H2O to MeOH gradients. This yielded procerolides A, C and D (1, 3-4) and

procerone A (5). Extraction of another collection of P. procera and successive HPLC yielded procerolide B (2) and procerone B (6).

O OR 2 2 2' Br O A 4 1 R1 = R2 = H (S) B 6" OH 2 R1 = Br, R2 = H R1 6 6' 3 R1 = H, R2 = Me C Br 4 R1 = Br, R2 = Me MeO 2" Br

O HO 1 2' Br (S) B R 6" 3 6' OH 5 R = H C 6 R = Br Br MeO 2" Br Figure 1. New butenolides: the procerolides (1 - 4) and new diphenylpropanones: the procerones (5 - 6), isolated from P. procera.

Table 1. NMR Spectroscopic Data for Procerolides A - D (1 - 4) in DMSO-d6. procerolide A (1) procerolide B (2) procerolide C (3) procerolide D (4) Position δC, type δH (J in Hz) δC, type δH (J in Hz) δC, type δH (J in Hz) δC, type δH (J in Hz) 2 168.5, C 168.4, C 167.0, C 167.1, C 3 138.4, C 138.3, C 139.9, C 140.0, C 3-OR2 58.4, CH3 3.84, s 58.7, CH3 3.77, s 4 126.6, C 126.9, C 135.8, C 136.3, C 5 77.4, CH 5.80, dd (5.9, 4.0) 77.2, CH 5.84, dd (6.1, 4.0) 77.0, CH 5.88, dd (6.3, 3.9) 77.0, CH 5.91, dd (7.3, 4.4) 6 37.6, CH2 2.79, dd (14.7, 6.0) 38.0, CH2 2.78, dd (14.7, 6.1) 37.4, CH2 2.79, dd (14.6, 6.3) 37.9, CH2 2.84, dd (14.4, 7.3) 3.14, dd (14.7, 4.0) 3.15, dd (14.7, 4.0) 3.11, dd (14.6, 3.9) 3.09, dd (14.4, 4.4) 1' 125.1, C 125.0, C 123.7, C 123.7, C 2' 131.0, CH 7.84, s 131.0, CH 7.86, s 131.5, CH 7.80, s 131.8, CH 7.80, s 3' 112.3, C 112.2, C 112.1, C 112.2, C 4' 151.0, C 150.9, C 151.9, C 152.1, C 5' 112.3, C 112.2, C 112.1, C 112.2, C 6' 131.0 CH 7.84, s 131.0, CH 7.86, s 131.5, CH 7.80, s 131.8, CH 7.80, s 1" 128.8, C 135.4, C 128.5, C 134.9, C 2" 133.9, CH 7.17, d (1.3) 133.9, CH 7.42, s 133.8, CH 7.18, d (1.7) 134.1, CH 7.40, s 3" 110.0, C 116.8, C 109.9, C 117.0, C 4" 154.3, C 152.1, C 154.2, C 152.3, C 4"-OMe 56.2, CH3 3.78, s 60.4, CH3 3.77, s 56.2, CH3 3.80, s 60.6, CH3 3.89, s 5" 112.2, CH 6.96, s 116.8, C 112.1, CH 6.99, s 117.0, C 6" 130.3, CH 6.96, d (1.3) 133.9, CH 7.42, s 130.2, CH 6.98, d (1.6) 134.1, CH 7.40, s 1H NMR was measured at 500 MHz for 1, 3, 4 and 800 MHz for 2. 13C NMR was measured 125 MHz for 1, 3, 4 and 200 MHz for 2.

Procerolide A (1), isolated as a pale yellow solid, had an ion cluster for [M - H]- at m/z

544.8243/546.8227/548.8209/550.8189 (1:3:3:1) in the (-) HRESIMS data, consistent with a

1 molecular formula of C18H13O5Br3. The H NMR data (Table 1) contained resonances associated with five aromatic protons (H 7.84 - 6.96), one oxygenated methine (H 5.80), one

13 O-methyl (H 3.78), and two geminal coupled aliphatic protons (H 3.14, 2.79). The C NMR

and HSQC data indicated the presence of ten non-protonated carbons (C 168.5, 154.3, 151.0,

138.4, 128.8, 126.6, 125.1, 112.3 (2C), 110.0), five aromatic methines (C 133.9, 131.0 (2C),

130.3, 112.2), an oxygenated methine (C 77.4), an O-methyl (C 56.2), and one aliphatic carbon (C 37.6). HMBC correlations from the methine at  5.80 (H-5) to carbons at C

168.5 (C-2), 138.4 (C-3), and 126.6 (C-4) allowed a 3,4,5-trisubstituted furan-2(5H)-one to be assigned (ring A). The deshielded resonance at C 138.4 (C-3) of the furanone ring indicated it was hydroxy substituted. A symmetrical 1,3,4,5-tetrasubstituted aromatic ring was identified from HMBC correlations from the 2H singlet at H 7.84 (H-2'/6'). The deshielded resonance of C-4' (C 151.0) suggested that it was hydroxylated and the chemical shift of the non-protonated carbons, C-3'/5' (C 112.3) were consistent with bromine

6 substitution. An HMBC correlation from H 7.84 (H-2'/6') to C 126.6 (C-4) indicated that the 3,5-dibromo-4-hydroxyphenyl moiety (ring B) is attached to C-4 of the furanone. A 1,3,4- trisubstituted aromatic ring was identified from HMBC correlations from H 6.96 (H-5") to C

128.8 and 110.0 (C-1" and C-3" respectively) and from H 7.17 (H-2") and 6.96 (H-6") to C

154.3 and 112.2 (C-4" and C-5" respectively). The HMBC correlation from  3.78 (C-4" O- methyl) to C 154.3 (C-4") and attachment of a bromine atom to the non-protonated carbon C-

3" allowed the assignment of the aromatic substituents. The methylene protons at H

2.79/3.14 (C 37.6) correlated to the furanone methine proton, H 5.80 (H-5) in the COSY spectrum, while the aromatic protons at H 7.17 and 6.96 (H-2" and H-6") showed an additional HMBC correlation to the aliphatic methylene carbon at C 37.5 (C-6). This indicated that the 3-bromo-4-methoxybenzyl moiety (ring C) is attached to C-5 of the furanone ring. With the 2D structure of 1 established, the absolute configuration of the stereogenic center, C-5, was determined through comparison of experimental and predicted electronic circular dichroism (ECD) spectra. ECD spectra of the two possible enantiomers of

1 were calculated using time-dependent density functional theory (TDDFT) using the

B3LYP/6-31G(d)//B3LYP/6-31G(d) functional/basis set combination. The calculated ECD spectrum of the (5S)-1 enantiomer matched with the experimental spectrum (Figure 2b).

Therefore, procerolide A was assigned as (S)-(+)-4-(3,5-dibromo-4-hydroxyphenyl)-5-(3- bromo-4-methoxybenzyl)-3-hydroxyfuran-2(5H)-one (1).

Procerolide B (2), isolated as a pale yellow solid, had an ion cluster for [M - H]- at m/z

622.7337/624.7314/626.7308/628.7299/630.7302 (1:4:6:4:1) in the (-) HRESIMS data,

1 13 consistent with a molecular formula of C18H12O5Br4. Comparison of H NMR and C NMR data between 1 and 2 indicated that ring C was tetrasubstituted in 2 instead of trisubstituted in

1. This was confirmed by an aromatic singlet at H 7.42 that integrated for two protons, indicating symmetry in the aromatic ring. This, in addition to MS data indicated an additional bromine was attached to C-5" in ring C. The absolute configuration of the C-5 stereogenic center was assigned through the comparison of optical rotation between 1 and 2. As both optical rotations were positive the C-5 stereogenic center was assigned in an S configuration.

Therefore, procerolide B was assigned as (S)-(+)-4-(3,5-dibromo-4-hydroxyphenyl)-5-(3,5- dibromo-4-methoxybenzyl)-3-hydroxyfuran-2(5H)-one (2).

Procerolide C (3), isolated as a pale yellow solid, had an ion cluster for [M - H]- at m/z

558.8386/560.5372/562.8354/564.8328 (1:3:3:1) in the (-) HRESIMS data, consistent with a

1 13 molecular formula of C19H15O5Br3. The H NMR and C NMR data for 3 contained signals for an additional O-Me group (H 3.84, C 58.4) compared to 1. An HMBC correlation between H 3.84 and C 139.9 (C-3) indicated the O-Me was attached to C-3 in ring A. Again the absolute configuration of the C-5 stereogenic center was assigned through the comparison of optical rotation between 1 and 3. A positive rotation indicated that the molecule had a 5S

configuration. Therefore, procerolide C was assigned as (S)-(+)-4-(3,5-dibromo-4- hydroxyphenyl)-5-(3-bromo-4-methoxybenzyl)-3-methoxyfuran-2(5H)-one (3).

Procerolide D (4), isolated as a pale yellow solid, had an ion cluster for [M - H]- at m/z

636.7508/638.7500/640.7496/642.7462/644.7434 (1:4:6:4:1) in the (-) HRESIMS data,

1 13 consistent with a molecular formula of C19H14O5Br4. Comparison of H NMR and C NMR data between 2 and 4 indicated an additional O-Me group (H 3.77, C 58.7). A HMBC correlation between H 3.77 and C 140.0 (C-3) indicated the O-Me was attached to C-3 in ring A. Similar to the previous compounds the positive optical rotation matching that observe for 1 allowed the assignment of a 5S configuration. Therefore, procerolide D was assigned as

(S)-(+)-4-(3,5-dibromo-4-hydroxyphenyl)-5-(3,5-dibromo-4-methoxybenzyl)-3- methoxyfuran-2(5H)-one (4).

(a) O OH

O Br

OH Br MeO Br 1 COSY HMBC

(b)

Figure 2: (a) Key COSY and HMBC correlations for 1. (b) Comparison of the experimental ECD spectrum of 1 and the calculated spectrum of the (S)- and (R)- enantiomers of 1 at the B3LYP/6-31G(d) level. The procerolides are butenolides related to the previously reported ascidian metabolites rubrolides,7 cadiolides8 and prunolides.6 The procerolides differ from the previous compounds because they have an additional degree of saturation at C-5 in the furanone ring.

The procerolides also differ in the substitution of C-3 in the furanone ring. The substitution at

C-3 of a hydroxyl or O-methyl has not been identified in related compounds from ascidians.

Ascidian-derived butenolides have been found to possess a wide range of bioactivities including, anti-bacterial,9, 10 anti-viral,11 and anti-inflammatory activity,12 photosynthesis inhibition,13 and cancer cell cytotoxicity.14 Similar butenolide metabolites with the additional degree of saturation at C-5 and hydroxyl substitution at C-3 have also been identified the

yeast Aspergillus terreus.15 These compounds were found to inhibit cyclin-dependent kinases which have important functions in a number of diseases including cancer, Alzheimer’s and

Parkinson’s.16

Table 2. NMR Spectroscopic Data for Procerones A - B (5 - 6) in DMSO-d6. procerone A (5) procerone B (6)

Position δC, type δH (J in Hz) δC, type δH (J in Hz) 1 197.2, C 197.0, C 2 73.2, CH 5.01, dd (8.2, 4.9) 72.5, CH 5.07, dd (8.6, 4.6) 2-OH 5.68, bs 3 38.3, CH2 2.77, dd (14.1, 8.2) 38.0, CH2 2.77, dd (14.0, 8.6) 2.93, dd (14.1, 4.9) 2.93, dd (14.0, 4.6) 1' 128.0, C 129.3, C 2' 133.2, CH 8.11, s 133.2, CH 8.10, s 3' 112.3, C 111.5, C 4' 155.0, C 155.1, C 4'-OH 10.98, bs 5' 112.3, C 111.5, C 6' 133.2, CH 8.11, s 133.2, CH 8.10, s 1" 131.7, C 137.8, C 2" 133.7, CH 7.45, d (2.2) 133.9, CH 7.52, s 3" 110.1, C 116.9, C 4" 153.9, C 151.7, C 4"-OMe 56.2, CH3 3.81, s 60.4, CH3 3.75, s 5" 111.5, CH 7.00, d (8.4) 116.9, C 6" 130.0, CH 7.18, dd (8.3, 2.2) 133.9, CH 7.52, s 1H NMR was measured at 500 MHz for 5 and 800 MHz for 6. 13C NMR was measured at 125 MHz for 5 and 200 MHz for 6.

Procerone A (5), isolated as a pale yellow solid, had an ion cluster for [M - H]- at m/z

504.8260/506.8259/508.8240/510.8198 (1:3:3:1) in the (-) HRESIMS data, consistent with a

1 molecular formula of C16H13O4Br3. The H NMR data for 1 and 5 (Table 2) were similar, however, comparison of 13C NMR and HSQC data for 1 and 5 showed that 5 has two fewer non-protonated olefinic carbon resonances and the ester carbonyl (C 168.3) is replaced with a ketone carbonyl (C 197.2). Comparison of 2D NMR data between 1 and 5 indicated that 5 also contained 3-bromo-4-methoxyphenyl (ring B) and 3,5-dibromo-4-hydroxyphenyl (ring

C) moieties. COSY correlations between an oxygenated methine at H 5.01 (H-2) and methylene protons at H 2.77/2.93 (H2-3) indicated they are attached to vicinal carbons.

HMBC correlations from H 5.01 and 2.77/2.93 (H-2 and H2-3) to the carbonyl resonance at

C 197.2 (C-1) as well as the further deshielding of H-2 (H 5.01) compared to typical oxygenated methine protons indicated the presence of the 2-hydroxypropanone moiety.

HMBC correlations from H 8.11 (H-2' and H-6') to C 197.2 (C-1) and from H 7.45 (H-2") and H 7.18 (H-6") to C 38.3 (C-3) indicated the attachment of rings B and C to the 2- hydroxypropanone. With the 2D structure of 5 established, the absolute configuration of the stereogenic center, C-2, was determined through comparison of experimental and predicted

ECD spectra as described previously. The calculated ECD spectrum of the (2S)-5 enantiomer had the closest match to the experimental spectrum (Figure 3d). This configuration was further supported from results obtained using the Mosher’s ester method.17 The esterification of the C-2 hydroxyl with the (R)- and (S)-MTPA acid chloride afforded the (S)- and (R)-

MTPA esters of 5, 5S and 5R, respectively. Analysis of the 1H NMR and HSQC data for these MTPA esters of 5 indicted the deshielding of H-2'/6' and the shielding of H-6", H-5" and H2-3 in 5S compared to 5R (Figure 3c). This indicated the position of the phenyl moiety of the MTPA in both 5S and 5R, allowing the assignment of a 2S configuration. Therefore, 5 was assigned as (S)-(+)-3-(3-bromo-4-methoxyphenyl)-1-(3,5-dibromo-4-hydroxyphenyl)-2- hydroxypropanone.

Procerone B (6), isolated as a pale yellow solid, had an ion cluster for [M - H]- at m/z

582.7405/584.7393/586.7378/588.7359/590.7337 (1:4:6:4:1) in the (-) HRESIMS data,

1 consistent with a molecular formula of C16H12O4Br4. The H NMR data for 5 was almost identical to that reported for 6 except that a 2H aromatic singlet at H 7.54 replaced the three ring C protons in 6. Ring C was therefore 1,3,4,5-tetrasubstituted and since the MS data indicated that 6 contains an additional bromine, C-5" must be brominated. Therefore, 6 was assigned as (S)-(+)-1-(3,5-dibromo-4-hydroxyphenyl)-3-(3,5-dibromo-4-methoxyphenyl)-2- hydroxypropanone.

We hypothesize that the procerones are likely derived from the procerolides. The procerones contain aromatic rings B and C linked via a 2-hydroxypropanone moiety instead of the

furanone moiety in the procerolides. The procerolides are most likely amino acid derived, through the enzymatic condensation of two alpha-keto acids derived from tyrosine.18 The decarboxylation and reduction at C-5 would then generate the procerolides (Figure 3a). We surmise that the procerones are biosynthesized through the cleavage of an acetic acid unit from the procerolides. These procerolides and procerones may then be precursors to a number of other larger butenolide and related metabolites.

H (a) HO O Br O O OH HO Br

Br OH O OH Br MeO MeO O Br 5

Br O

HO enzymatic cleavage

HO O Br O OH O OH Br O O O 1. decarboxylation Br OH HO 2. keto-enol tautomerism Br MeO MeO Br 1 Br (b) (c) O O +0.04 HO Br RO Br -0.21 OH -0.10 -0.02 OH Br -0.02 Br MeO MeO Br Br 6 6R: R = (R)-MTPA ester COSY HMBC 6S: R = (S)-MTPA ester

(d)

Figure 3: (a) The proposed biosynthetic pathway of the procerolides and then to the procerones. (b) Key COSY and HMBC correlations for 5. (c) Δδ values [Δδ (in ppm) = δS – δR] obtained for (S)- and (R)-MTPA esters of 5. (d) Comparison of the experimental ECD spectrum of 5 and the calculated spectrum of the (S)- and (R)- enantiomers of 5 at the B3LYP/6-31G(d) level.

The procerolides and procerones exhibited significant spectroscopic differences dependent on the basicity of solutions. Under basic conditions the 1H NMR of procerolide A showed both the shielding and broadening of the resonances associated with H-2'/6' and H-5 compared to under acidic conditions (Figure 4a). Additionally, the 13C NMR resonances associated with

C-2, C-3, C-4, C-1', C-3'/5' and C-4' were not visible, and those associated with C-5, C-6, C-

2'/6' and C-1" had significantly reduced intensities. In the UV spectrum of procerolide A a bathochromic shift (λmax 288→353) was observed under basic conditions (Figure 4c).

Similarly, in the 1H NMR of procerone B the shielding and broadening of the resonances associated with H-2'/6' and H-5 (Figure 4b) along with a bathochromic shift correlated with a change in the basicity. This spectroscopic data indicates that the procerolides and the procerones undergo keto-enol tautomerism under basic conditions. We propose that this tautomerism in the procerolides is the interconversion of the phenol-hydroxyfuranone moiety with a dienone-dihydroxyfuranylidene moiety (Figure 4a) and in the procerones the interconversion of the phenol-hydroxypropanone moiety with a dienone- dihydroxypropylidene moiety (Figure 4b).

(a) O HO OH OH +0.08 O Br O Br +0.10 R R OH O Br Br base (b) O OH +0.02 HO Br HO Br +0.03 R R OH O Br Br base (c)

base

Figure 4: The proposed keto-enol tautomerism within the procerolides, (a), and the procerones, (b), under basic conditions with Δδ values of protons [Δδ (in ppm) = δacidic – δbasic]. (c) The observed bathochromic that accompanied the tautomerism of both 1 and 5. The procerolides and procerones were screened for anti-prion activity using yeast containing either the [PSI+] or the [URE3] prions (Table 3).5 Guanabenz, a potent anti-prion compound, was used as a positive control for a comparison of activity. Guanabenz has been shown to cure yeast prions at concentrations <30 μM and also inhibit mammalian prions.19 Procerolide

A - B (1 - 2) and procerones A - B (5 - 6) all cured the yeast prions at a comparable dose to guanabenz. Since both the procerolides and the procerones are active against yeast prions, this indicates that the rings B and C are likely to be important for anti-prion activity. A number of previously reported anti-prion compounds have two phenyl moieties connected via a hydrophilic moiety, in a similar arrangement to that present in the procerolides and procerones. These include anle138b,20 and N-benzylidene-benzohydrazide.21 Procerolides C and D (3 and 4) have significantly weaker anti-prion activity compared to 1, 2, 5 and 6.

Procerolides C and D (3 and 4) are methoxylated at C-3 and therefore lack a hydrogen-bond donor moiety between rings B and C. This hydrogen bond donor may be important for anti-

prion activity. It is also important to note that previous studies, with similar butenolide metabolites, have suggested that the differences in anti-microbial activity is likely to reflect differences in a compounds ability to permeate cell membranes.22 Since activity in the in vivo yeast-based assay used in this study is dependent on the ability of the compounds to pass through the cell membrane, the membrane permeability of the compounds can influence the potency of their anti-prion activity. The calculated LogP of the procerolides and procerones

(Table 3) indicated that their permeability may influence their EC50’s. The results obtained in this study support the view that the procerolides and the procerones may help in the future design and development of novel lead compounds for neurodegenerative diseases.

Table 3: Anti-prion activity of the procerolides (1-4) and the procerones (5-6). ClogP [PSI+] Prion Curing [URE3] Prion Curing Compounds EC50 ± SE (μM) Curing at 100 μM Procerolide A (1) 5.12 22.7 ± 9.0 Yes Procerolide B (2) 5.76 34.2 ± 3.2 Yes Procerolide C (3) 5.75 64.6 ± 4.2 Yes Procerolide D (4) 6.21 67.1 ± 5.4 Yes Procerone A (5) 4.61 29.4 ± 4.8 Yes Procerone B (6) 5.20 33.2 ± 6.7 Yes Guanabenz 1.69 26.0 ± 4.5 Yes

EXPERIMENTAL SECTION

General Experimental Procedures. UV spectra were measured using a Shimadzu UV-1800

UV spectrophotometer. IR spectra were measured using a Thermo Scientific Nicolet iS5 iD5

ATR spectrometer. CD spectra were measured using a JASCO J-715 spectropolarimeter.

Optical rotations were measured using a JASCO P-1020 polarimeter. NMR spectra were recorded at 25 °C on a Bruker Avance III 500 MHz spectrometer (BBFO Smartprobe, 5mm

31P-109Ag) and a Bruker BioSpin GmbH 800 MHz spectrometer with a triple (TCl) resonance 5 mm cryoprobe. NMR solvent DMSO-d6 peak was referenced to H 2.50 and C

39.52. High resolution ESI-TOF data was recorded on an Agilent 6530-accurate mass Q-

TOF LC/MS mass spectrometer with a 1200 Series autosampler and 1290 Infinity HPLC.

Altech Davisil 35-76 m 60 Å C18 silica was used to adsorb samples prior to HPLC separation. A Merck Hitachi L7100 pump and a L7455 PDA detector were used for HPLC.

HPLC columns used were a Thermo Betasil C18 5 m, 100 Å, 150 mm x 21.2 mm and a

Phenomenex EVO C18 5 m, 100 Å, 150 mm x 21.2 mm. All solvents used for chromatography and MS were HPLC grade and the H2O was Millipore Milli-Q PF filtered.

Trifluoroacetic acid (TFA) was spectroscopy grade from Alfa Aesar. Sodium chloride (NaCl) was analytical grade from Merck. Analytical grade compounds, guandine hydrochloride

(GuHCl) and guanabenz were purchased from Sigma. The S. cerevisiae STRg6 strain was used to screen against the [PSI+] prion (MATa ade1-14 trp1-289 his3Δ200 ura3-52 leu2-

3,112 erg6::TRP1 [PSI+]). The S. cerevisiae SB34 strain was used to screen against the

[URE3] prion (MATa ade2-1 trp1-1 leu2-3,112 his3-11,15 ura2::HIS3 dal5::ADE2 [URE3]).

Greiner sterile 96-well plates were used for screening.

Animal Material. Samples of the ascidian Polycarpa procera were collected on June 21,

2011, by hand using scuba in the shallow waters off the coast of Coffs Harbour, NSW,

Australia. This sample was freeze-dried and stored at room temperature. A voucher specimen

(ACENV0263) is located at Griffith University, Gold Coast, Queensland, Australia. The ascidian was taxonomically identified by A.R.C.

Extraction and Isolation. The freeze-dried sample of the ascidian P. procera (5.41 g) was exhaustively extracted with MeOH to yield a crude extract (0.2930 g). This extract was adsorbed onto the C18 silica gel at a 1:1 ratio and packed into the HPLC refillable cartridge

(10 mm x 20 mm) that was connected in series with a Betasil C18 bonded silica HPLC column (21 mm x 150 mm). The columns were eluted with a gradient from 100% H2O containing 0.1% TFA to 100% MeOH containing 0.1% TFA over 60 min at a flow rate of 9 mL/min. The column was further eluted with 100% MeOH containing 0.1% TFA for 10 min.

A total of 70 fractions were collected at one min intervals. 0.45 mL aliquots (5% of each fraction) from seven sequential fractions were combined yielding ten fractions for anti-prion testing. Active fractions were analysed by 1H NMR; fractions 43 to 46 were recombined for further separation (28 mg), fraction 47 contained procerolide C (3, 2.8 mg, 0.052% dry wt) and fraction 50 contained procerolide D (4, 6.8 mg, 0.13% dry wt). Fractions 44-46 were separated using a Betasil C18 bonded silica HPLC column (21 mm x 150 mm). The column was eluted with a gradient from 80% H2O containing 0.1% TFA/20% MeOH containing

0.1% TFA to 40% H2O containing 0.1% TFA/60% MeOH containing 0.1% TFA over 60 min, then to 100% MeOH containing 0.1% TFA over 5 min at a flow rate of 9 mL/min. The column was then further eluted with 100% MeOH containing 0.1% TFA for 10 min. A total of 75 fractions were collected at one min intervals. Fraction 24 and 25 contained procerone A

(5, 0.4 mg, 7.4 x 10-3 % dry wt) and fractions 30 and 31 contained procerolide A (1, 1.2 mg,

0.022% dry wt.).

Another sample of the freeze-dried ascidian P. procera (44.58 g) was exhaustively extracted with MeOH yielding another crude extract (4.98 g). This extract was adsorbed onto the C18 silica gel at a 1:1 ratio and separated on a C18 silica flash column using a H2O/MeOH gradient. Four fractions were collected by eluting 80% H2O/20% MeOH, 20% H2O/80%

MeOH, 10% H2O/90% MeOH and 100% MeOH. The 20% H2O/80% MeOH fraction (1.2 g) was adsorbed onto the C18 silica gel at a 1:1 ratio and packed into the HPLC refillable cartridge (10 mm x 20 mm) that was connected in series with a Betasil C18 bonded silica

HPLC column (21 mm x 150 mm). The columns were eluted with a gradient from 80% H2O containing 0.01 M NaCl/20% MeOH to 20% H2O containing 0.01 M NaCl/80% MeOH over

60 min, then to 100% MeOH over 5 min at a flow rate of 9 mL/min. The column was then further eluted with 100% MeOH for 10 min. A total of 75 fractions were collected at one min intervals. Fractions 20 to 35 were recombined (90 mg) and separated using an EVO C18

bonded silica HPLC column (21 mm x 150 mm). The column was eluted with a gradient from 80% H2O containing 0.01 M NaCl/20% MeOH to 20% H2O containing 0.01 M

NaCl/80% MeOH over 60 min, then to 100% MeOH over 5 min at a flow rate of 9 mL/min.

The column was then further eluted with 100% MeOH for 10 min. A total of 75 fractions were collected at one min intervals. Fractions 39 to 42 (6.5 mg) and fractions 59-60 (7.8 mg) were recombined for further separation. Fractions 39-42 were separated using an EVO C18 bonded silica HPLC column (21 mm x 150 mm). The column was eluted with a gradient from 40% H2O containing 0.1% TFA/60% MeOH containing 0.1% TFA to 20% H2O containing 0.1% TFA /70% MeOH containing 0.1% TFA over 60 min, then to 100% MeOH containing 0.1% TFA over 5 min at a flow rate of 9 mL/min. The column was then further eluted with 100% MeOH containing 0.1% TFA for 10 min. A total of 75 fractions were collected at one min intervals. Fractions 49 and 50 contained procerone B (6, 0.15 mg, 3.4 x

-4 10 % dry wt.). Fractions 59-60 were separated using an EVO C18 bonded silica HPLC column (21 mm x 150 mm). The column was eluted with a gradient from 50% H2O containing 0.1% TFA/50% MeOH containing 0.1% TFA to 100% MeOH containing 0.1%

TFA over 60 min, at a flow rate of 9 mL/min. The column was then further eluted with 100%

MeOH containing 0.1% TFA for 10 min. A total of 70 fractions were collected at one min intervals. Fraction 47 contained procerolide B (2, 1.4 mg, 0.026% dry wt.).

25 Procerolide A (1): pale yellow amorphous solid; [α]D +12 (c 0.05, MeOH); UV/Vis λmax

MeOH (log ε) 289 nm (3.86), 229 nm (4.24); ECD (c 0.1 mM) λmax MeOH (Δε) 368 (0.00),

288 (+2.63), 266 (0.00), 257 (-0.79), 249 (0.00), 239 (+1.02), 234 (0.00), 213 (-6.14) nm; IR

-1 1 13 (film) νmax 3323, 2944, 2832, 1662, 1449, 1416, 1116, 1021 cm ; H NMR and C NMR see

Table 1. (-) HRESIMS m/z [M - H]- 544.8243/546.8227/548.8209/550.8189 (1:3:3:1) (calcd

- for C18H12O5Br3 544.8235).

25 Procerolide B (2): pale yellow amorphous solid; [α]D +21 (c 0.1, MeOH); UV λmax MeOH

(log ε) 289 nm (3.74), 227 nm (4.25); IR (film) νmax 3324, 2945, 2833, 1662, 1449, 1417,

1116, 1020cm-1; 1H NMR and 13C NMR see Table 1; (-) HRESIMS m/z [M - H]-

622.7337/624.7314/626.7308/628.7299/630.7302 (1:4:6:4:1) (exact mass calcd for

- C18H11O5Br4 626.7340).

25 Procerolide C (3): pale yellow amorphous solid; [α]D +19 (c 0.1, MeOH); UV λmax MeOH

(log ε) 290 nm (3.83), 221 nm (4.18); IR (film) νmax 3324, 2945, 2833, 1647, 1449, 1116,

1020 cm-1; 1H NMR and 13C NMR see Table 1; (-) HRESIMS m/z [M - H]-

- 558.8386/560.5372/562.8354/564.8328 (1:3:3:1) (calcd for C19H14O5Br3 558.8391).

25 Procerolide D (4): pale yellow amorphous solid; [α]D +30 (c 0.1, MeOH); UV λmax MeOH

(log ε) 299 nm (3.89), 221 nm (4.06); IR (film) νmax 3323, 2943, 2832, 1646, 1449, 1417,

1116, 1022 cm-1; 1H NMR and 13C NMR see Table 1; (-) HRESIMS m/z [M - H]-

- 636.7508/638.7500/640.7496/642.7462/644.7434 (1:4:6:4:1) (calcd for C19H13O5Br4

636.7497).

25 Procerone A (5): pale yellow amorphous solid; [α]D +15 (c 0.1, MeOH); UV λmax MeOH

(log ε) 279 nm (3.42), 220 nm (3.93); ECD (c 0.025 mM) λmax MeOH (Δε) 291 (+3.34), 249

(+0.85), 241 (0.00), 217 (-4.41) nm; IR (film) νmax 3324, 2944, 2832, 1647, 1449, 1417, 1116,

1021 cm-1; 1H NMR and 13C NMR see Table 2; (-) HRESIMS m/z [M - H]-

- 504.8260/506.8259/508.8240/510.8198 (1:3:3:1) (calcd for C16H12O4Br3 504.8286).

25 Procerone B (6): pale yellow amorphous solid; [α]D +10 (c 0.1, MeOH); UV λmax MeOH

(log ε) 285 nm (3.83), 221 nm (4.19); IR (film) νmax 3324, 2944, 2832, 1646, 1449, 1116,

1021 cm-1; 1H NMR and 13C NMR see Table 2; (-) HRESIMS m/z [M - H]-

- 582.7405/584.7393/586.7378/588.7359/590.7337 (1:4:6:4:1) (calcd for C16H11O4Br4

582.7391).

Computational Methods. The computational methods used for the calculation of ECD and

UV spectra are as described previously.23 Generation of conformers was carried out using

Schrodinger MacroModel 2016 using parameters outlined by Willoughby et al.24 Subsequent calculations based on density functional theory (DFT) were then performed on each of the conformers to optimise the structures at the B3LYP/6-31G(d) level using Gaussian 16.25

Zero-point energy calculations, electronic transition and rational strength were calculated at the same level. The polarisable continuum model was used during all calculations.26

Boltzmann-weighted UV and ECD spectra were calculated using the freely available software SpecDis 1.7.27

Preparation of the MTPA Esters of Procerone A (5). The method used was based on the methods previously described by Hoye et al.17 Deuterated pyridine (0.78 μL, 9.80 μmol, 10 equiv.) was added to the procerone (5, 0.5 mg, 0.98 μmol) in dry deuterated chloroform (300

μL, 3.3 mM) in a 5 mm NMR tube. The S-(+)-α-methoxy-α-trifluoromethylphenylacetic acid chloride (S-(+)-MTPA-Cl) (0.74 μL, 3.92 μmol, 4 equiv.) was added at room temperature.

The reaction progress was monitored by 1H NMR. After the completion (~30 mins) the reaction mixture containing the R-MTPA-5 ester (5R) was analysed by 1H NMR and HSQC.

5R was present in a 1:1 ratio with 5 indicating a 50% completion of the reaction. 5R 1H NMR

(CDCl3) 8.02 (s, H-2'/6'), 7.16 (dd, J = 2.2, 8.3 Hz, H-6"), 6.86 (d, J = 8.3 Hz, H-5"), 5.19

(t, J = 6.1 Hz, H-2), 3.86 (O-methyl), 3.07 (dd, J = 5.9, 14.1, H-3a), 2.94 (dd, J = 6.3, 14.1,

H-3b). Using analogous methodology as described above the S-MTPA-7 ester was prepared by using (±)-MTPA-Cl. The reaction mixture containing the S-MTPA-7 ester (7S) was

1 1 analysed by H NMR and HSQC. 5S was present in a 1:1 ratio with 5R. 5S H NMR (CDCl3)

8.06 (s, H-2'/6'), 6.95 (dd, J = 2.3, 8.4 Hz, H-6"), 6.76 (d, J = 8.4 Hz, H-5"), 5.19 (t, J = 6.2

Hz, H-2), 3.86 (O-methyl), 3.04 (dd, J = 6.0, 14.1 H-3a), 2.92 (dd, 6.2, 14.2 H-3b).

In Vivo Yeast-Based Anti-Prion Assay. The anti-prion activity was determined using the method reported by Jennings et al.5 In summary, yeast harbouring the [PSI+] or [URE3] prion were incubated in the presence of a compound or extract. [PSI+] is the prion form of the

Sup35 protein that has a critical role in translation termination in yeast. The STRg6

Saccharomyces cerevisiae strain was used for testing against the [PSI+] prion. [URE3] is the prion form of the Ure2 protein that is important for controlling of expression of genes for nitrogen metabolism in yeast. The SB34 S. cerevisiae strain was used for testing against the

[URE3] prion. Cultures of STRg6 and SB34 are white when Sup35p and Ure2p are in their infectious and insoluble prion form and red when they are in their normal non-infectious and soluble form. Assays were performed in 96-well plates with a working volume of 250 μL or in 384-well plates with a working volume of 50 μL. Plates were incubated for 48 hrs at 24°C to allow the yeast to grow and then for a further 48 hrs at 4°C for the colour to develop.

Images were taken with a Canon CanoScan LiDE 220 flatbed colour scanner and red colour intensity was analysed using ImageJ 1.51j8. The red colour intensity was also quantified by measuring fluorescence (ex. 544nm/em. 620nm) on a BMG Labtech FLUOstar Omega microplate fluorimeter. The percent curing of the prion was calculated by comparison of the colour intensity of samples with that of DMSO (0% curing) and 100 μM guanabenz (100% curing) controls. Dose-response curves were plotted and the IC50 value for each compound was calculated.

ASSOCIATED CONTENT

Supporting Information. Electronic supplementary information is available free of charge on the ACS Publications website at DOI:

1D and 2D NMR spectra for procerolides A-D (1-4) and procerones A-B (5-6). 1D

and 2D NMR spectra for procerolide A (1) and procerone B (6) under basic

conditions.

AUTHOR INFORMATION

Corresponding author

*Tel: +61 7 55529187. Fax: +61 7 55529047. E-mail: [email protected].

Orcid

Laurence K. Jennings 0000-0002-1313-0360

Luke P Robertson 0000-0001-5987-2426

Alan L. Munn 0000-0002-4085-9405

Anthony R. Carroll 0000-0001-7695-8301

Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENT

We thank CNRS in Roscoff, France for providing the yeast strains used in the assay. The authors acknowledge the Griffith University eResearch Services for the use of the Computing

Cluster “Gowonda” to calculate ECD spectra. We thank W. Loa-Kum-Cheung, and J.

Carrington for their technical assistance. LKJ was supported by an Australian Government

Research Training Program Scholarship.

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CHAPTER 3:

New Anti-prion and α-synuclein Aggregation Inhibitory Sterols from the Australian Ascidian, Didemnum sp.

STATEMENT OF CONTRIBUTION: This section includes a co-authored research manuscript that has been prepared for submission to Journal of Natural Products; “New Anti-prion and α-synuclein Aggregation Inhibitory Sterols from the Australian Ascidian, Didemnum sp.” The details of the co-authored manuscript, including all authors, is:

Jennings, L. K.; Prebble, D. W.; Xu, M.; Munn, A. L.; Mellick, G. D.; Carroll, A. R. New Anti-prion and α-synuclein Aggregation Inhibitory Sterols from the Australian Ascidian, Didemnum sp. J. Nat. Prod. In preparation for submission

My contribution to the paper involved physical experimentation and organization of information, data and references, as well as the preparation of the manuscript.

(Signed)______

Laurence Kane Jennings Date:______

(Signed)______

Corresponding author: Anthony R. Carroll Date:______

(Signed)______

Supervisor: Anthony R. Carroll Date:______

JOURNAL OF NATURAL PRODUCTS MANUSCRIPT:

New Anti-prion and α-synuclein Aggregation Inhibitory Sterols from the Australian Ascidian, Didemnum sp.

Laurence K. Jennings,†,‡ Dale W. Prebble,†,‡ Mingming Xu,‡,§ Alan L. Munn,┴ George D.

Mellick,‡,§ Anthony R. Carroll*,†,‡,§

†Environmental Futures Research Institute, Griffith University (Gold Coast campus),

Parklands Drive, Southport, QLD 4222, AUSTRALIA.

‡School of Environment and Science, Griffith University (Gold Coast campus), Parklands

Drive, Southport, QLD 4222, AUSTRALIA.

§Griffith Institute for Drug Discovery, Griffith University (Brisbane Innovation Park), Don

Young Road, Nathan, QLD 4111, AUSTRALIA.

┴School of Medical Science and Molecular Basis of Disease Program, Menzies Health

Institute Queensland, Griffith University (Gold Coast campus), Parklands Drive, Southport,

QLD 4222, AUSTRALIA.

*[email protected]

ABSTRACT: In a recent bio-discovery study with the aim of identifying new anti-prion compounds we screened a library of 500 Australian marine invertebrate derived extracts using a yeast-based anti-prion assay. This resulted in an extract from the subtropical ascidian

Didemnum sp. showing potent anti-prion activity. The bioassay-guided chemical investigation of this ascidian extract led to the isolation of three new poly-oxygenated steroids, the didemnisterols (1-3). These sterols were all isolated in low yield and the structures of 1-3 were elucidated from extensive NMR and MS analysis. Compounds 1-3

+ displayed potent anti-prion activity against the [PSI ] yeast prion (EC50s of 12.7 μM, 13.8 μM and 9.8 μM, respectively). The major component 1 was then evaluated further and was found to bind to α-synuclein and to significantly inhibit the aggregation of amyloid fibrils of α- synuclein. Our findings indicate that low yielding sulfated poly-oxygenated steroids may be useful anti-neurodegenerative compounds.

The build-up of toxic misfolded proteins is thought to contribute to the development and progression of a number of neurodegenerative diseases. These misfolded proteins typically contain a large proportion of β-sheets and this facilitates their association into toxic, soluble oligomers and then their aggregation into insoluble amyloid fibrils.1 Neurodegenerative diseases and their associated amyloid proteins include: Alzheimer’s disease with tau and β- amyloid proteins,2 Parkinson’s disease and Multiple Systems Atrophy with the α-synuclein protein,3 Amyotrophic Lateral Sclerosis with the FUS, TDP-43 and SOD1 proteins,4

Huntington’s disease with the Huntingtin protein,5 and Creutzfeldt–Jakob disease and Fatal

Familial Insomnia with the prion protein (PrP).6 Generally, the amyloid aggregation cascade leads to the gradual loss of neuronal cells and eventually death. There are few available treatment options for patients suffering from these diseases and no current drugs directly target their associated misfolded proteins.7, 8

Recently, research has begun to focus on the development of drugs that target both the misfolding and aggregation of disease-implicated amyloid proteins.7, 8 Since similar proteins that have the capacity to misfold and aggregate have been identified in a variety of organisms,9-11 we hypothesized that natural products may exist that can regulate or inhibit the formation of protein aggregates in diverse organisms. High-throughput biological assays have been developed to screen natural extracts to identify compounds that modulate protein misfolding. These assays include in vitro mass-spectrometry based protein binding and thioflavin-T based fluorescence staining assays, and in vivo yeast-based anti-prion assays.12-14

These assays have facilitated the discovery of natural products with anti-neurodegenerative disease activity.

Marine organisms are a diverse source of bioactive compounds and a number of unique drug leads have been developed that are based on marine natural products.15 We have screened a library of 500 extracts derived from sub-tropical Australian marine macro-organisms using a

yeast-based anti-prion assay.14 This led to the identification of an extract from the ascidian

Didemnum sp. with anti-prion activity. Herein, we report the isolation and identification of a series of new bioactive sulfated poly-oxygenated sterols. These compounds cured prion infected yeast cells in vivo, and inhibited α-synuclein aggregation in vitro.

RESULTS AND DISCUSSION

We have recently developed in vivo assays that allow for the identification of direct and indirect activity associated with prion mechanisms at the misfolding, aggregation or propagation stages of prion formation for two yeast amyloid prions, [PSI+] and [URE3].14

These anti-prion assays were used to screen a large collection of Australian sub-tropical marine macro-organism extracts, and this resulted in an extract from the ascidian, Didimnum sp. being identified to possess potent anti-prion activity. Exhaustive extraction of the freeze- dried ascidian with MeOH was followed by preparative C18 silica gel HPLC separation eluting with a gradient from H2O to MeOH. Fractions were screened for anti-prion activity and subsequent semi-preparative HPLC and bioassay driven isolation yielded three new anti- prion sterols, didemnisterols A-C (1-3).

18 R

11 H 19 H 16 1 9 D C 14 H OH 3 A 5 B 7 HO OH H

22 24 26 1: R = OSO3H

2: R = (S) OSO3H 28

3: R = OSO3H

Figure 1. New anti-prion sterols, didemnisterols (1-3), isolated from Didemnum sp.

Table 1. NMR spectroscopic data for 1-3 in DMSO-d6 Didemnisterol A (1) Didemnisterol B (2) Didemnisterol C (3) Position δC, Type δH (J in Hz) δC, Type δH (J in Hz) δC, Type δH (J in Hz) α 1.19, m 1.19, m 1.19, m 1 32.6, CH2 32.7, CH2 32.7, CH2 β 1.27, m 1.28, m 1.29, m α 1.56, m 1.55, m 1.57, m 2 28.4, CH2 28.4, CH2 28.4, CH2 β 1.43, m 1.43, m 1.43, m 3 β 64.3, CH 3.79, q (2.7) 64.0, CH 3.79, q (2.7) 63.9, CH 3.79, q (2.2) 3 α-OH 4.14, d (2.8) 4.13, d (2.8) 4.10, m α 1.08, m 1.09, m 1.09, m 4 35.4, CH2 35.4, CH2 35.1, CH2 β 1.34, td (13.2, 2.7) 1.34, td (13.2, 2.7) 1.34, td (13.2, 2.7) 5 α 30.7, CH 2.11, tt (13.1, 2.5) 30.6, CH 2.11, tt (13.1, 2.5) 30.6, CH 2.11, tt (13.3, 2.9) α 0.90, m 0.90, m 0.90, m 6 32.5, CH2 31.9, CH2 32.2, CH2 β 1.71, td (13.2, 2.4) 1.71, td (13.2, 2.4) 1.71, td (13.3, 1.8) 7 β 70.6, CH 3.28, under H2O 70.2, CH 3.29, under H2O 70.3, CH 3.30, under H2O 7 α-OH 4.30, d (4.6) 4.29, d (4.6) 4.29, d (4.3) 8 75.2, C n.d., C n.d., C 8 β-OH 3.19, s 3.21, s 3.21, s 9 α 49.2, CH 1.11, m 49.0, CH 1.12, m 49.2, CH 1.12, m 10 35.5, C 36.5, C 35.9, C α 1.38, m 1.38, m 1.41, m 11 17.5, CH2 17.4, CH2 17.1, CH2 β 1.59, m 1.59, m 1.59, m α 1.06, m 1.06, m 1.09, m 12 40.4, CH2 40.6, CH2 40.7, CH2 β 1.88, dt (12.3, 3.2) 1.86, dt (12.3, 3.2) 1.86, dt (12.6, 3.1) 13 42.6, C 43.4, C 42.8, C 14 α 53.2, CH 1.54, dd (13.0, 7.0) 52.9, CH 1.54, m 53.1, CH 1.55, dd (13.0, 7.0) α 1.46, m 1.45, m 1.44, m 15 17.9, CH2 17.8, CH2 17.7, CH2 β 1.37, m 1.38, m 1.38, m α 1.14, m 1.26, m 1.27, m 16 27.5, CH2 27.8, CH2 27.9, CH2 β 1.74, m 1.57, m 1.58, m 17 α 56.4, CH 0.97, m 55.9, CH 1.02, q (9.6) 56.0, CH 1.01, q (9.6) 18 13.6, CH3 0.87, s 13.6, CH3 0.89, s 13.7, CH3 0.89, s 19 11.5, CH3 0.87, s 11.5, CH3 0.87, s 11.6, CH3 0.87, s 1.96, ddd (9.7, 7.8, 20 34.6, CH 1.31, m 39.0, CH 39.2, CH 1.94, dt (9.6, 7.3) 6.7) 21 18.3, CH3 0.85, d (6.6) 20.3, CH3 0.94, d (6.7) 20.5, CH3 0.94, d (6.7) 1.28, m 22 35.9, CH2 138.3, CH 5.20, dd (15.1, 7.9) 135.0, CH 5.16, dd (15.3, 7.8) 0.98, m 1.26, m 5.24, ddd (15.1, 23 22.7, CH2 124.8, CH 131.5, CH 5.20, dd (15.3, 7.1) 1.19, m 7.1, 6.0) 1.25, m 2.00, dt (13.1, 5.6) 24 33.4, CH2 35.8, CH2 37.4, CH 2.08, m 1.02, m 1.69, m 25 33.0, CH 1.61, m 33.2, CH 1.66, m 37.6, CH 1.62, m 3.54, dd (9.5, 5.9) 3.55, dd (9.7, 6.2) 3.68, dd (9.6, 5.9) 26 70.6, CH2 70.0, CH2 68.4, CH2 3.46, dd (9.5, 6.8) 3.45, dd (9.7, 6.9) 3.40, dd (9.6, 8.0) 27 16.8, CH3 0.82, d (6.6) 16.1, CH3 0.80, d (6.6) 13.4, CH3 0.76, d (6.6) 28 22.6, CH3 0.86, d (6.6) 1 13 H NMR was measured at 800 MHz for 1 - 3 and C NMR was measured at 200 MHz for 1. δC shifts for 2 and 3 were derived from analysis of HSQC and HMBC data measured at 800 MHz.

Didemnisterol A (1), isolated as a clear amorphous solid, had a deprotonated molecule [M -

- H] at m/z 515.3035 in the (-) HRESIMS, consistent with a molecular formula of C27H48O7S.

Analysis of the 1H NMR and HSQC data (Table 1) indicated 1 contained resonances associated with three hydroxyl protons (H 4.30, 4.14, 3.19), two oxygenated methine protons

(H 3.79, 3.28), two oxygenated methylene protons (H 3.54, 3.46), six methine protons (H

2.11, 1.61, 1.54, 1.31, 1.11, 0.97), 22 methylene protons (H 0.90 - 1.87), two coincident

13 methyl singlets (H 0.87) and two methyl doublets (H 0.85, 0.82). The C NMR and HSQC data (Table 1) indicated that 1 contained resonances associated with one non-protonated oxygenated carbon (C 75.2), two oxygenated methines (C 70.6, 64.3), one oxygenated methylene (C 70.6), four bridge head methines (C 56.4, 53.2, 49.2, 30.7), two non- protonated aliphatic carbons (C 42.6, 35.5), eleven aliphatic methylenes (C 40.4, 35.9, 35.4,

33.4, 32.6, 32.5, 28.4, 27.5, 22.7, 17.9, 17.5), two aliphatic methines (C 34.6, 33.0), and four methyls (C 18.3, 16.8, 13.6, 11.5). HMBC correlations from the coincident methyl resonances at H 0.87 to two quaternary, four methine and two methylene carbon resonances at C 56.4, 53.2, 49.2, 42.6, 40.4, 35.5, 32.6, 30.7 indicated the presence of two bridge head

16 methyls characteristic of C-18 and C-19 in steroids. HMBC correlations from H 2.11,

1.27/1.19 and 1.11 (H-5, H2-1, and H-9, respectively) to C 11.5 (C-19), and from H

1.88/1.06, 1.54 and 0.97 (H2-12, H-14, and H-17, respectively) to C 13.6 (C-18) allowed for the separation of the HMBC correlations from the two coincident methyls so that the two non-protonated bridge head carbons, C-10 and C-13, could be assigned. COSY correlations from H 2.11 (H-5) to H 1.71/0.90 (H2-6) and 1.34/1.08 (H2-4) indicated two methylenes were vicinal to the C-5 bridge head. HMBC correlations from H-5 to oxygenated carbons at

C 70.6 and 64.3 indicated that these carbons could be assigned to C-7 and C-3, respectively, both of which are three bonds away from the bridge head proton. HMBC correlations between the hydroxy proton at H 4.14 (3-OH) and C 64.3, 35.4 and 28.4 (C-3, C-4 and C-2 respectively), as well as a correlation from H 3.79 (H-3) to C 32.6 (C-1), allowed ring A to be assigned. The hydroxy proton at H 4.30 (7-OH) COSY correlated to an oxygenated methine at H 3.28 and showed a HMBC correlation to C 75.2 (C-8) indicating that the oxygenated methine (C-7) is vicinal to an oxygenated quaternary carbon (C-8). HMBC

correlations between the hydroxy proton singlet at H 3.19 (8-OH) and C 75.2, 53.2 and 49.2

(C-8, C-14 and C-9) allowed ring B to be assigned. Intense COSY correlations from H 1.59

(H-11β) to H 1.06 (H-12α) and H 1.11 (H-9), along with an HMBC correlation from H2-12 and H-9 to C 17.5 (C-11), established the presence of ring C. COSY correlations from H

1.37 (H-15β) to H 1.74 and 1.54 (H-16β and H-14), as well as from H-16β to H 0.97 (H-17), allowed the assignment of ring D. Further COSY and HMBC correlations from H 0.97 (H-

17) indicated an acyclic chain was attached to C-17. A HMBC correlation from the secondary methyl resonance at H 0.85 (H3-21) to C 56.4 (C-17) and a COSY correlation from H3-21 to

H 1.31 (H-20) indicated that a CH3CH was directly attached to C-17 of the steroid. HMBC correlations from the secondary methyl resonance at H 0.82 (H3-27) to C 70.6, 33.4 and

33.0 (C-26, C-24 and C-25, respectively) indicated that C-26 was an oxygenated methylene.

Intense TOCSY correlations from the methyl doublets at H 0.85 and 0.82 (H3-21 and H3-27 respectively) to signals between H 1.20-1.30 that were associated with three unassigned methylenes indicated a propyl linked to the two secondary methyl moieties in the chain. No additional COSY correlations were observed from the oxygenated methylene protons at H

3.54/3.46 (H2-26), suggesting that the sulfate group was attached to C-26. With the 2D structure of 1 established, ROESY correlations were used to assign the relative configuration of nine of the ten stereogenic centres in the molecule (Fig. 2). Correlations between H-5 and the hydroxy protons (H 4.30 (7-OH), 4.14 (3-OH)) and H-9 indicated the α axial configuration of these groups. A ROSEY correlation from the hydroxy proton at H 3.19 to the methyl protons H3-18 and H3-19 indicated their β axial configurations. The relative configuration of the steroid side chain was assigned to the β face of the molecule from

ROSEY correlations between H 1.54 (H-14) and H 4.30 (7-OH) and 0.97 (H-17). A large

COSY correlation between H-17 and H-20 and the lack of ROESY correlations between

these protons suggested that they are in an anti-conformation. A ROESY correlation between the methyl protons H 0.85 (H3-21) and 1.88 (H-12β) then indicated that C-21 is equatorial and close to C-12. The configuration of the last stereogenic centre (C-25) was left unassigned due to a lack of spectral data required for its assignment. Therefore, didemnisterol A was assigned as 3α,7α,8β-trihydroxy-5α-cholestane-26-sulfate.

(a)

OH OSO3H

HO OH

COSY HMBC (b)

Figure 2: (a) Selected COSY and HMBC correlations of 1-3. (b) Chem3D energy minimised structure of 1 with selected ROESY correlations. Didemnisterol B (2), isolated as a clear amorphous solid, had a deprotonated molecule [M -

- H] at m/z 513.2885 in the (-) HRESIMS, consistent with a molecular formula of C27H46O7S.

Comparison of the 1H NMR and 2D NMR data (Table 1) between 1 and 2 indicated differences only in the aliphatic chain of the steroid, with two of the methylene resonances being replaced with two olefinic resonances. COSY correlations between H 5.24 and 5.20

(H-22 and H-23) and a large homonuclear coupling (J = 15.1 Hz) indicated the presence of a trans-double bond. This double bond is vicinal to C-20 because a COSY correlation was observed between H-20 and H-22. This assignment was further confirmed by the observation

of an HMBC correlation from H 0.95 (H-21) to C 138.3 (C-22). The same configuration as 1 was assigned based on the similarity of the spectral data. However, assignment of C-25 was possible as a recent paper has synthesised compounds containing 26-sulfated cholest-22-ene

(Fig. 3 (4)) derivatives with either a 25R or 25S configuration. This paper reported a subtle difference in the proton and carbon chemical shifts for the methylene groups on either side of

17 1 this stereogenic centre. Analysis of the H NMR and COSY of 2 in D2O indicated that the

H2-24 (H 2.04/1.95) methylene protons matched the reported shifts of the 25S configuration

(Δ25S–Exp. = -0.02/-0.01 and Δ25R–Exp. = 0.04/0.09). This allowed the assignment of 25S configuration in 2. Therefore, didemnisterol B was assigned as (22E, 25S)-3α,7α,8β- trihydroxy-5α-cholest-22-ene-26-sulfate. The configuration assigned to C-25 in 2, suggests that the configuration at C-25 in 1 is most likely the same.

Didemnisterol C (3), isolated as a clear amorphous solid, had a deprotonated molecule [M -

- H] at m/z 527.3024 in the (-) HRESIMS, consistent with a molecular formula of C28H48O7S.

Comparison of the 1H NMR and 2D NMR data between 2 and 3 indicated differences in the steroid acyclic chain. The 1H NMR data for 3 (Table 3) had an additional methyl doublet observed at H 0.86 (H-28). In addition, the doublet of doublet of doublet multiplicity for H-

23 in 2 (ddd, J = 15.3, 7.0, 6.0 Hz) was replaced in 3 with a doublet of doublets (dd, J = 15.3,

7.2 Hz), indicating that C-23 is vicinal to a methine instead of a methylene in 3. HMBC correlations from the H3-28 to C 131.5, 38.6 and 38.4 (C-23, C-24 and C-25 respectively) substantiated the assignment of its position. Based on the spectroscopic data, the same relative configuration of the steroid ring carbons and C-20 as that assigned in 1 and 2 was assigned in 3. However, due to a lack of unambiguous NMR correlative data the, stereogenic centres, C-24 and C-25 would not be assigned. Therefore, didemnisterol C was assigned as

(22E)-24-methyl-3α,7α,8β-trihydroxy-5α-cholest-22-ene-26-sulfate.

The didemnisterols are related to the previously reported sulfated sterols from Ascidia sydneiensis (4),18 Ciona intestinalis (5)19 and Phallusia mamillata (6-8).20, 21 Both 4 and 5 have been identified as sperm-activating and attracting factors (SAAF)18 and 6-8 have been found to induce pregnane X receptor (PXR) transactivation.21 The Ascidia-SAAF (4) is the most similar sulfated sterol to the didemnisterols, being the only compound to contain a C-8 hydroxy. The C-8 hydroxy was reported to be a genus-specific sperm attractant. The structural differences between the didemnisterols (1-3) and 4 is the absence of a sulfate group at C-3 and differences in the side chains in 1 and 3. Interestingly, the didemnisterols were isolated in low yields, similar to those of the previously isolated sulfated ascidian steroids.

This may indicate that only low concentrations of these compounds are required for their biological roles.

H H H OSO3Na H

H OH H H NaO3SO OH HO H 4 NaO SO 3 OH 6: 6R 7: 6S

H H OSO3Na H OSO3Na H

H H H H NaO SO OH NaO SO 3 H 3 OH 5 8 Figure 3: Previously isolated sulfated steroids from ascidians: Ascidia-SAAF (4),18 Ciona-SAAF (5),19 phallusiasterols A-C (6-8).20, 21 Didemnisterols A-C (1-3) were evaluated for anti-prion activity using the yeast-based anti- prion bioassays targeting either the [PSI+] or [URE3] prions (Table 2). Didemnisterols A-C

+ (1-3) were found to cure both of the yeast prions with EC50s for the [PSI ] curing of 12.7 μM,

13.8 μM and 9.8 μM, respectively. These are significantly lower than that of guanabenz, an anti-prion compound shown to have potent bioactivity against both yeast and mammalian prions.22

Table 2: Anti-prion and α-synuclein aggregation inhibitory activity of the didemnisterols (1-3) compared to other known anti-prion and α-synuclein aggregation inhibiting compounds. [PSI+] [URE3] α-synuclein ThT Prion Curing Prion Curing Fluorescence Inhibition Compounds EC50 ± SE (μM) Yes/No at 40 μM % ± SE (5 :1 mole ratio) didemnisterol A (1) 12.7 ± 1.4 Yes 68.0 ± 2.9 didemnisterol B (2) 13.8 ± 3.5 n.d.* n.d.* didemnisterol C (3) 9.8 ± 5.1a n.d.* n.d.* epigallocatechin gallate - - 96.3 ± 0.2 guanabenz 26.0 ± 4.5 Yes - *Didemnisterols B and C (2, 3) were not screened against α-synuclein or [URE3] prion. aDidemnisterol C (3) was screened against the [PSI+] prion with only two replicates.

To further evaluate the bioactivity of 1, it was tested for its ability to bind to α-synuclein in vitro and inhibit its aggregation in vitro using MS and thioflavin T (ThT) assays respectively.

α-Synuclein aggregation was tested because of suggestions that the propagation of α- synucleinopathy in Multiple Systems Atrophy and other forms of Parkinsonism may have mechanistic similarities to those observed for transmissible-prions.23, 24 Didemnisterol A (1) was incubated with α-synuclein at a 5:1 molar ratio (1 : α-syn) for 3 hours and a mass spectrum was acquired for the resulting solution. Additional charged peaks in the spectra indicated the formation of a molecular complex between α-synuclein and 1 at a 1:1 molar ratio (Fig. 3). To evaluate the effect of the observed binding on α-synuclein aggregation, 1 was incubated at a 5:1 molar ratio with α-synuclein for 72 hours and thioflavin-T was used to quantify amyloid formation. The addition of didemmnisterol A (1) resulted in a significant reduction (~70%) in the ThT fluorescence compared to the untreated α-synuclein control indicating a significant reduction in the formation of amyloid by α-synuclein (Table 2). The direct nature of the inhibition of α-synuclein aggregation by didemnisterol A (1) suggests that the yeast prion inhibition may also be due to a direct interaction between didemnisterol A (1) and the yeast prions, thereby inhibiting their aggregation.

Figure 4: MS spectrums from the binding assays: (a) The mass spectrum of untreated α-synuclein, with arrows indicating the charge state of each peak. (b) The mass spectrum of α-synuclein treated with didemnisterol A, with arrows indicating the additional peaks attributed to a 1:1 α-synuclein-didemnisterol complex. It has been shown that a number of cholesterol-based human sex steroids, including estrogens, androgens, and progesterones, have important neuroprotective activity following brain injury.25 The same studies also indicated that these sex steroids have neuroprotective effects against Alzheimer’s disease, Parkinson’s disease and Motor Neurone Disease with the initiation of steroid receptor signalling in the brain thought to be strongly associated with a decrease in neuronal apoptosis.26 This is especially interesting, since the didemnisterols are similar to previously identified ascidian metabolites that are important for reproductive signalling. Recently, the ability to elucidate structures of compounds isolated in low yield has improved with better technology. Therefore, we suggest that low yielding sulfated steroids produced by ascidians and other marine organisms be examined for their activity against neurodegenerative disorders including those cause by prions and α-synuclein.

EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured using a JASCO P-

1020 polarimeter. NMR spectra were recorded at 25 °C on a Bruker BioSpin GmbH 800

MHz spectrometer equipped with a triple (TCl) resonance 5 mm cryoprobe. The NMR solvent DMSO-d6 peaks were referenced to H 2.50 and C 39.52 and NMR solvent D2O was referenced to the residual MeOH peak at H 3.34. A Bruker maXis II ETD Q-TOF LC/MS mass spectrometer was used to obtain high resolution ESI-TOF data and to analyze protein- ligand interactions in the MS bioassay. Altech Davisil 35-76 m 60 Å C18 silica was used to adsorb sample prior to HPLC separation. A Merck Hitachi L7100 pump and L7455 PDA detector were used for HPLC. HPLC columns used were a Thermo Betasil C18 5 m, 100 Å,

150 mm x 21.2 mm; and a Thermo Betasil C18 5 m, 100 Å, 250 mm x 10 mm. All solvents used for chromatography and MS were HPLC grade and the H2O was Millipore Milli-Q PF filtered. Trifluoroacetic acid (TFA) was spectroscopy grade from Alfa Aesar. Analytical grade compounds, guandine hydrochloride (GuHCl) and guanabenz were purchased from

Sigma. The S. cerevisiae STRg6 strain was used to screen for anti-prion activity using the

[PSI+] prion (MATa ade1-14 trp1-289 his3Δ200 ura3-52 leu2-3,112 erg6::TRP1 [PSI+]). The

S. cerevisiae SB34 strain was used to screen for anti-prion activity using the [URE3] prion

(MATa ade2-1 trp1-1 leu2-3,112 his3-11,15 ura2::HIS3 dal5::ADE2 [URE3]). Greiner sterile 96-well plates were used for screening. A BMG Labtech FLUO star Omega microplate fluorimeter was used to measure fluorescence and absorbance in the yeast-based anti-prion assay. A BioTek Synergy 2 microplate reader was used to measure fluorescence in the ThT assays.

Animal Material. The ascidian Didemnum sp. was collected on June 21, 2011, by hand using scuba at the offshore Reefs at Sawtell Shoal, Coffs Harbour, NSW, Australia. This sample was freeze-dried and stored at room temperature. A voucher specimen (ACENV0260) is

located at Griffith University, Gold Coast, Queensland, Australia. The taxonomy was performed by A.C.

Extraction and Isolation. The freeze-dried sample of the ascidian Didemnum sp. (5.53 g) was exhaustively extracted with MeOH to yield a crude extract (0.40 g). This extract was adsorbed onto the C18 silica gel at a 1:1 ratio and packed into a HPLC refillable cartridge (10 mm x 20 mm) that was connected in series with a Betasil C18 bonded silica HPLC column

(21 mm x 150 mm). The columns were eluted with a gradient from 100% H2O containing

0.1% TFA to 100% MeOH containing 0.1% TFA over 60 min at a flow rate of 9 mL/min.

The column was further eluted with 100% MeOH containing 0.1% TFA for 10 min. A total of 70 fractions were collected at one min intervals. 0.20 mL aliquots (2% from each fraction) from seven sequential fractions were combined yielding ten fractions for anti-prion testing.

Active fractions were analysed by 1H NMR and fractions 41 to 47 were recombined (1.0 mg) for further separation. Fractions 41-47 were separated using a Betasil C18 bonded silica HPLC column (10 mm x 250 mm). The column was eluted with a gradient from 50% H2O/50%

MeOH to 100% MeOH over 60 min at a flow rate of 9 mL/min. The column was then further eluted with 100% MeOH for 10 min. A total of 70 fractions were collected at one min intervals. Fraction 26 contained didemnisterol B (2, 0.08 mg, 1.45 x 10-3 % dry wt), fraction

28 contained didemnisterol A (1, 0.15 mg, 2.71 x 10-3 % dry wt) and fraction 30 contained didemnisterol C (3, 0.05 mg, 9.04 x 10-4 % dry wt).

25 1 13 Didemnisterol A (1). Clear amorphous solid; [α] D +0.3 (c 0.011, MeOH); H NMR and C

- - NMR see Table 1; (-) HRESIMS m/z [M - H] 515.3035 (calcd for C27H47O7S 515.3048).

1 13 Didemnisterol B (2). Clear amorphous solid; H NMR and C NMR (DMSO-d6) see Table

1 1; H NMR (800 MHz, D2O) 5.39 (m, 2H), 4.04 (brs, 1H), 3.93 (dd, J = 9.5, 6.2 Hz, 1H),

3.85 (dd, J = 9.5, 6.6 Hz, 1H), 3.56 (brs, 1H), 2.08-1.98 (m, 3H), 1.94-1.85 (m, 5H), 1.82-

100

1.72 (m, 3H), 1.66 (m, 1H), 1.60-1.10 (m, 12H), 0.99 (d, J = 6.6 Hz, 3H), 0.92 (d, J = 6.5 Hz,

- - 3H), 0.89 (s, 3H), 0.88 (s, 3H); (-) HRESIMS m/z [M - H] 513.2886 (calcd for C27H45O7S

513.2891).

Didemnisterol C (3). Clear amorphous solid; 1H NMR and 13C NMR see Table 1; (-)

- - HRESIMS m/z [M - H] 527.3024 (calcd for C28H47O7S 527.3048).

In vivo Yeast-Based Anti-Prion Assay. Saccharomyces cerevisiae strains harbouring the

[PSI+] or [URE3] prions were used for the anti-prion screening using published methodology.14 The STRg6 S. cerevisiae strain was used for anti-prion tests using [PSI+], the prion form of the Sup35p associated with translation termination. The SB34 S. cerevisiae strain was used for anti-prion tests using [URE3], the prion form of the Ure2p associated with nitrogen metabolism. The yeast is white when the protein of interest (Sup35p or Ure2p) is in its infectious prion form and the yeast is red when the protein of interest is in its normal non- infectious, soluble form. Assays were performed in 96-well plates with a working volume of

250 μL. Plates were incubated for 48 hours at 24°C for growth and then for a further 48 hours at 4°C for colour development. The red colour was quantified by absorption using a BMG

Labtech FLUO star omega microplate reader. The percentage curing of the yeast was calculated by comparison with the infected and uninfected controls. Dose response curves were plotted and the EC50 value was calculated for each compound.

Expression and purification of α-synuclein. Escherichia coli BL21 (DE3) cells containing the human α-synuclein (α-syn) gene (SNCA) inserted into a pET-22b (+) vector (Novagen,

Merck, MA, USA) were induced with 1 mM isopropyl β-D-1-thiogalactopyranoside (Sigma

Aldrich, MO, USA). α-Syn was first purified using the non-chromatographic protocol reported by Volles and Lansbury.27 Further purification using anion exchange was based on the protocol reported by Pujols et al.28

101

In vitro α-synuclein Binding MS Assay. An α-syn protein solution (10 μM) was prepared in

10 mM ammonium acetate. Pure compounds (50 μM) were dissolved in 100% MeOH and incubated with α-syn for 3 hours at room temperature. The protein-ligand interactions were then analyzed using (+) HRESIMS with the following parameters: end plate offset 500 V; capillary 4500 V; nebulizer 2.0 Bar; dry gas 7 L/min; dry temp 150°C; funnel I RF 400 Vpp;

ISCID 0.0 ev; ion energy 4.0 eV; collision energy 4.0 eV; transfer time 100 μs; multipole RF

800 Vpp; collision RF 1200 Vpp; prepulse storage 10 μs; spectra rate: 2×100 Hz. The spectrum obtained was analysed for additive peaks consistent with the exact mass of the compound. Sodium trifluoroacetate (0.1 mg/ml) was used to calibrate the instrument.

In vitro Fluorescence-based ThT α-synuclein Aggregation Assay. A stock solution (5 mM) of thioflavin T (ThT) was prepared in glycine-NaOH buffer (pH 8.0). To obtain consistent results, homogenous monomeric α-syn was made using the commonly used protocol described by Rahimi et al.29 The α-syn monomers (80 μM) were resuspended in aggregation buffer (20 mM Tris-HCl, pH 7.4, 150 mM NaCl and 0.05% NaN3) and incubated at 37 °C with constant shaking (1000 rpm in a thermomixer) for 72 hours with and without compounds (400 μM). For each treatment group, pre-incubated α-syn samples (60 μL) were mixed with a ThT solution (240 μL, 50 μM). The 300 μL solution was dispensed in triplicate into the wells of a 96-well plate and the plates were analyzed using a fluorimeter (ex. 440 nm/em. 500 nm). The fluorescence intensity of the samples was compared to the negative control that was untreated and positive control that was treated with epigallocatechin gallate to obtain percentage inhibition.

ASSOCIATED CONTENT

Supporting Information. Electronic supplementary information is available free of charge at the ACS Publications website at DOI:

102

1D and 2D NMR spectra of the didemnisterol mixture and purified didemnisterols A-

C (1-3).

AUTHOR INFORMATION

Corresponding Author

*Tel: +61 7 55529187. Fax: +61 7 55529047. E-mail: [email protected]

Orcid

Laurence K. Jennings 0000-0002-1313-0360

Dale W. Prebble 0000-0002-7084-8299

Mingming Xu 0000-0002-8158-2879

Alan L. Munn 0000-0002-4085-9405

George D. Mellick 0000-0002-7211-4651

Anthony R. Carroll 0000-0001-7695-8301

Notes

The authors have no competing financial interest to declare.

ACKNOWLEDGMENT

We thank Marc Blondel, Cecile Voisser, and Flavie Soubigou, CNRS in Roscoff, France for providing the yeast strains used in this study. We thank W. Loa-Kum-Cheung for technical assistance. LKJ was supported by an Australian Government Research Training Program

Scholarship. We also thank Griffith University for funding the Q-TOF MS through a Griffith

University Research Infrastructure Program (GURIP).

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CHAPTER 4:

Poly-oxygenated sterols from the sponge Dysidea sp. that exhibit potent inhibitory activity against yeast prions

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ABSTRACT The methanol extract from the sponge Dysidea sp. collected from subtropical coastal waters off the east coast of Australia was active in a yeast-based anti-prion bioassay. Bioassay guided chemical investigation of the specimen resulted in the isolation of a series of known poly-oxygenated steroids with anti-prion activity. Extensive 1D/2D NMR and MS analysis led to the structures of four acetylated Dysidea sterols (1-4) being elucidated. The acetate (1-2) and diacetate (3-4) sterols were then evaluated for their ability to cure yeast prion infections. The compounds displayed potent yeast-based anti-prion activity (EC50’s of 29.4 μM and 30.1 μM, respectively). This study has further highlighted that poly-oxygenated sterols are a new class of anti-prion compounds and that other marine organisms, where there is a precedent for the presence of similar poly- oxygenated steroids, should be targeted for structure-activity relationships.

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INTRODUCTION Prion diseases are fatal neurodegenerative disorders that are caused by the switching of a protein in its normal, soluble form into an infectious misfolded protein form.1 These misfolded proteins then associate, forming toxic oligomers and amyloids in the brain. Currently, no treatment options are available that either inhibit or slow the progression of these diseases.2 We therefore used a yeast-based anti-prion assay to screen a library of natural product extracts for anti-prion activity with the aim of identifying lead compounds that could be useful in the future design and development of novel anti- prion therapeutics.3 A screen of 500 marine invertebrate extracts using strains of the yeast, Saccharomyces cerevisiae, that are infected with either the [PSI+] or [URE3] prions, resulted in an extract from the sponge Dysidea sp. being found to possess bioactivity against both yeast prions. This extract was then analysed to identify the chemicals responsible for this anti-prion activity.

Herein, this chapter reports the isolation and structure elucidation of four known poly- oxygenated sterols from an Australian sponge, Dysidea sp. The anti-prion activity of these poly-oxygenated sterols was evaluated using a yeast-based anti-prion assay.

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RESULTS AND DISCUSSION A freeze-dried sample of the sponge Dysidea sp. was exhaustively extracted with

MeOH. The extract was separated by preparative gradient reverse-phase HPLC on C18 silica gel using a gradient from H2O to MeOH and the fractions were evaluated for anti- prion activity. The bioactive fractions were further separated by gradient normal-phase HPLC (Fig. 4-2). This yielded four known poly-oxygenated sterols, the Dysidea-sterols (1-4), in two mixtures. Our fractionation failed to separate the acyclic sterol chains. This separation was complicated by the low yields of these compounds.

Figure 4-1: Isolation diagram of the poly-oxygenated Dysidea-sterols (1-4) with anti-prion activity.

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Structure elucidation of Dysidea-sterol A-11-acetate (1) Dysidea sterol A-11-acetate (1) could only be isolated as a mixture with its C-22 olefin derivative 5 (1:1 molar ratio). A weak sodiated molecule [M+Na]+ was observed at m/z

547.3235 in the (+) HRESIMS, consistent with a molecular formula of C29H48O8. The structure of 1 was elucidated from extensive 1D and 2D NMR analysis. 21 22 18 24 AcO H 26 HO 17 19 11OH 13 HO 1 9 15 H HO 3 5 7 OH OH 1 Figure 4-2: Structure of Dysidea-sterol A-11-acetate (1).

Table 4-1: NMR data for Dysidea-sterol A-11-acetate (1) in DMSO-d6.

Pos. δC, Type δH (J in Hz) COSY HMBC ROESY

1 α 33.6, CH2 1.92, t (13.0) 1β, 2 2, 10, 19 3 β 1.78, dd (13.3, 4.7) 1α, 2 2, 3, 5, 10 2, 19 2 β 71.2, CH 3.30, m (under H2O) 1α, 1β, 3 3 1β, 4β, 19 3 α 71.0, CH 3.53, m 2, 4α, 4β 1α, 4α 4 α 38.6, CH2 1.54, dd (14.2, 5.4) 3, 4β 2, 3, 5, 6, 10 3 β 1.93, dd (14.3, 11.6) 3, 4α 3, 6, 10 19 5 76.3, C 6 α 70.0, CH 3.53, dd (5.7, 2.3) 7, 14 5, 7, 8, 10 7 7 121.2, CH 5.19, dd (5.8, 2.3) 6, 14 5, 9, 14 6, 15β, 15α 8 140.4, C 9 74.2, C 10 47.5, C 11 β 73.6, CH 5.34, dd (11.5, 4.7) 12α, 12β 11-OAc 12β, 18, 19b 12 α 42.1, CH2 1.57, m 11, 12β 11, 13, 17, 18 14, 17 β 1.96, m 11, 12α 9, 11, 14 11, 18, 21 13 42.3, C 14 α 50.3, CH 2.49, m 6, 7, 15β, 15α 7, 8, 12, 13, 15 12α, 15α, 16α, 17 15 α 22.7, CH2 1.45, dt (13.2, 6.6) 14, 15β, 16β 13 7, 14,16β β 1.34, m 14, 15α, 16β 14 7, 16β, 18 16 α 27.5, CH2 1.25, m 16β 17, 18, 20 18, 14 β 1.85, ddd (12.7, 6.4, 3.2) 15α, 15β, 16α 15α, 15β 17 α 55.3, CH 1.23, m 20, 16β 13, 16, 18, 20 12α, 14 18 12.2, CH3 0.66, s 12, 13, 14, 17 11, 12β, 15α, 16α, 20 19 a 63.5, CH2 3.66, d (13.7) 19b 5, 9, 10 2, 4β b 3.29, d (13.7) 19a 1, 5, 9, 10 1β, 11 20 35.6, CH 1.31, m 17, 21, 22 18 21 18.6, CH3 0.85, d (6.7) 20 17, 20, 22 12β 22 35.6, CH2 0.97, m 20, 23 0.96, m 20, 23 23 23.3, CH2 1.29, m 22, 24 1.11, m 22, 24 24 39.0, CH2 1.13, m 23, 25 1.08, m 23, 25 25 27.5, CH 1.50, m 24, 26, 27 26 22.5, CH3 0.84, d (6.6) 25 24, 25, 27 27 22.5, CH3 0.84, d (6.6) 25 24, 25, 26 11-OAc 169.5, C 21.8, CH3 1.99, s 11-OAc

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The 1H NMR and HSQC data (Table 4-1, Fig. 4-4) contained resonances associated with four oxygenated methines (H 5.34, 3.53 (2H), 3.30), one double bond proton (H

5.19), a geminally coupled oxygenated methylene (H 3.66, 3.29), two bridge head methines (H 2.49, 1.23), one acetate methyl (H 1.99), eight aliphatic methylenes (H

1.96 - 0.96), two aliphatic methines (H 1.50, 1.31), one methyl doublet (H 0.85), an

13 isopropyl methyl signal (H 0.84) and one methyl singlet (H 0.66). The C NMR and

HSQC data (Table 4-1) indicated the presence of one acetate carbonyl (C 169.5), one non-protonated double bond carbon (C 140.4), one double bond methine (C 121.2), two oxygenated non-protonated carbons (C 76.3, 74.2), four oxygenated methines (C

73.6, 71.2, 71.0, 70.0), one oxygenated methylene (C 63.5), two bridge head methines

(C 55.3, 50.3), two non-protonated carbons (C 47.5, 42.3), eight aliphatic methylenes

(C 42.1, 39.0, 38.6, 35.6, 33.6, 27.5, 23.3, 22.7), two aliphatic methines (C 35.6, 27.5), two isopropyl methyls (C 22.5 (2C)), one acetate methyl carbon (C 21.8), one secondary methyl (C 18.6), and one tertiary methyl (C 12.2).

Figure 4-3: 1H NMR spectrum of Dysidea-sterol A-11-acetate (1) and Dysidea-sterol B-11-acetate (2) in 1:1 mixture

in DMSO-d6.

HMBC correlations from the methyl resonance at H 0.66 to one quaternary, two methine and one methylene carbon resonances atC 42.3, 55.3, 50.2 and 42.1, respectively, were characteristic of carbons two and three bonds away from the C-18 111

methyl of a steroid. The NMR data, however, lacked a characteristic C-19 methyl singlet. An oxygenated methylene at H 3.66/3.29 and C 63.5 (CH2-19) indicated the presence of a CH2OH. Four HMBC correlations from H2-19 to C 76.3, 74.2, 47.5 and

33.6 indicated that the CH2OH was attached to a non-protonated carbon. This allowed the two quaternary carbons, C-10 and C-13, to be assigned. COSY correlations between

H 1.92/1.78 (H2-1) and 3.30 (H-2), H 3.30 (H-2) and 3.52 (H-3), and H 3.53 (H-3) and

1.93/1.54 (H2-4) allowed the assignment of two oxygenated methines at C-2 and C-3. A

HMBC correlation from H 1.54 (H-4α) to C 76.3 (C-5) allowed ring A to be assigned.

A COSY correlation between a coincident oxygenated methine at H 3.53 (H-6) and a double bond methine at H 5.19 (H-7), indicated that the two methines are vicinal. The

HMBC correlations from H 3.53 (H-6) to C 121.2 and 140.4 allowed the assignment of a non-protonated double bond carbon at C-8. Further HMBC correlations from H 5.19

(H-7) to the two oxygenated non-protonated carbons, C 76.3 and 74.2 (C-5 and C-9), allowed the assignment of ring B. Another HMBC correlation from H 5.19 (H-7) to C

50.2 (C-14) as well as a small allylic COSY correlation to H 2.49 (H-14) allowed the quaternary carbon, C-13, to be assigned two bonds from the olefinic C-8 carbon. A strong COSY correlation between H 1.57 (H-12α) and H 5.34 (H-11) and a HMBC correlation from H 1.57 (H-12α) to C 74.2 (C-9) allowed an oxygenated methine at C-

11, and ring C to be assigned. A weak HMBC correlation from H 5.34 to C 169.5, in addition to the further deshielding of the proton resonance allowed the assignment of the acetyl group on C-11. COSY correlations from H 1.45 and 1.34 (H2-15) to H 2.49 and 1.85 (H-14 and H-16β, respectively), in addition to a HMBC correlation from H

1.23 (H-17) to C 27.5 (C-16) allowed ring D to be assigned. Further spectroscopic data indicated the presence of an acyclic chain attached to C-17 of the steroid. A COSY correlation from H 0.85 (H3-21) to H 1.31 (H-20), and HMBC correlations from H

0.85 to C 55.3 (C-17) and 35.6 (C-20 and 22), indicated that CH3CH was directly attached to C-17. HMBC correlations from the methyl resonances at H 0.84 (H3-26/27) to C 39.0, 27.5 and 22.5 (C-25, C-24 and C-26/27) indicated an isopropyl attached to

C-24. COSY correlations from the methylene proton resonances at H 0.97/0.96 (H2-

22), and H 1.13/1.08 (H2-24) to a methylene at H 1.29/1.11 (H2-23) allowed the assignment of a propyl linker between the isopropyl and the CH3CH.

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AcO HO OH D HO A B HO OH OH COSY HMBC Figure 4-4: Key COSY and HMBC correlations used to elucidate of the chemical structure of 1. With the 2D structure of 1 established, ROESY correlations were used to assign the relative configuration. ROESY correlations from H 3.30 (H-2) to H 1.78 (H-1β) and

1.93 (H-4β) in addition to the strong COSY correlation to H 1.92 (H-1α) allowed the C- 2 hydroxy to be assigned to an α equatorial configuration. Similarly, ROESY correlations from H 3.52 (H-3) to H 1.92 (H-1α) and 1.54 (H-4α) as well as the strong

COSY correlation to H 1.93 (H-4β) allowed the C-3 hydroxy to be assigned in a β equatorial configuration. A ROESY correlation from H 3.30 (H-2) and 1.93 (H-4β) to

H 3.66/3.29 (H2-19) indicated that the C-19 methylene was in a β axial configuration. This also suggested that the C-5 hydroxy was in an α axial arrangement. ROESY correlations from H 5.34 (H-11) to H2-19 and H 1.96 (H-12β) as well as its large

homonuclear coupling (J = 11.5 Hz) with H 1.57 (H-12α) allowed the C-11 acetate to be assigned to an α equatorial configuration. This then indicated that the C-9 hydroxy was in an α axial configuration on ring C. A ROESY correlation between H 0.66 (H3-

18) and H 5.34 (H-11) indicated that the C-18 methyl was in a β axial arrangement. The

ROESY correlation between H 2.49 (H-14) and H 1.23 (H-17) indicated that the acyclic chain was in a β configuration. The ROESY correlations between the methyl at

H 0.85 (H3-21) and the C-12 equatorial proton at H 1.96 (H-12β), as well as between the methyl atH 0.66 (H3-18) and H 1.31 (H-20), allowed the assignment of the configuration of C-20. Therefore, Dysidea-sterol A-11-acetate was assigned as 2α,3β,5α,6β,9α,19-hexahydroxycholest-7-ene-11α-acetate.

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Figure 4-5: Key ROESY correlations used to assign the relative configuration of 1.

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Structure elucidation of Dysidea-sterol B-11-acetate (2) Dysidea-sterol B-11-acetate (2) could only be isolated as a mixture with 1 (1:1 molar ratio). A weak sodiated molecule [M+Na]+ was observed at m/z 559.3238 in the (+)

HRESIMS, consistent with a molecular formula of C30H48O8. The structure of 2 was elucidated from NMR analysis and by analogy with 1.

21 28 22 18 24 AcO H 26 HO 17 19 11OH 13 HO 1 9 15 H HO 3 5 7 OH OH 2 Figure 4-6: Structure of Dysidea-sterol B-11-acetate (2).

Table 4-2: NMR data for Dysidea-sterol B-11-acetate (2) in DMSO-d6.

Pos. δC, Type δH (J in Hz) COSY HMBC ROESY 1 α 33.6, CH2 1.92, t (13.0) 1β, 2 2, 10, 19 3 β 1.78, dd (13.3, 4.7) 1α, 2 2, 3, 5, 10 2, 19 2 β 71.2, CH 3.30, m (under H2O) 1α, 1β, 3 3 1β, 4β, 19 3 α 71.0, CH 3.53, m 2, 4α, 4β 1α, 4α 4 α 38.6, CH2 1.54, dd (14.2, 5.4) 3, 4β 2, 3, 5, 6, 10 3 β 1.93, dd (14.3, 11.6) 3, 4β 2, 3, 5, 6, 10 3 5 76.3, C 6 α 70.0, CH 3.53, dd (5.7, 2.3) 7, 14 5, 7, 8, 10 7 7 121.2, CH 5.19, dd (5.8, 2.3) 6, 14 5, 9, 14 6, 15β, 15α 8 140.4, C 9 74.2, C 10 47.5, C 11 β 73.6, CH 5.34, dd (11.5, 4.7) 12α, 12β 11-OAc 12β, 18, 19 12 α 42.1, CH2 1.57, m 11, 12β 11, 13, 17, 18 14, 17 β 1.96, m 11, 12α 9, 11, 14 11, 18, 21 13 42.3, C 14 α 50.2, CH 2.47, m 6, 7, 15β, 15α 7, 8, 12, 13, 15 12α, 15α, 16α, 17 15 α 22.7, CH2 1.45, dt (13.2, 6.6) 14, 15β, 16β 13 7, 14,16β β 1.34, m 14, 15α, 16β 14 7, 16β, 18 16 α 27.5, CH2 1.25, m 16β 17, 18, 20 18, 14 β 1.69, ddd (12.7, 6.4, 3.2) 15α, 15β, 16α 15α, 15β 17 α 54.9, CH 1.29, m 20, 16β 13, 16, 18, 20 12α, 14 18 12.4, CH3 0.68, s 12, 13, 14, 17 11, 12β, 15α, 16α, 20 19 63.0, CH2 3.66, d (13.7) 19 5, 9, 10 2, 4β 3.29, d (13.7) 19 1, 5, 9, 10 1β, 11 20 39.9, CH 1.96, m 21 18 21 20.9, CH3 0.94, d (6.70) 20 17, 20, 22 12β 22 135.5, CH 5.14, dd (15.3, 8.5) 20, 23 23 131.8, CH 5.22, dd (15.3, 7.7) 22, 24 24 42.5, CH 1.83, h 23, 25, 28 25 32.7, CH 1.44, m 24, 26, 27 26 19.5, CH3 0.79, d (6.8) 25 24, 25, 27 27 20.1, CH3 0.82, d (6.6) 25 24, 25, 26 28 18.0, CH3 0.88, d (6.9) 24 23, 24, 25 11-Oac 169.5, C 20.9, CH3 1.99, s 11-OAc

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Comparison of the NMR data (Table 4-2) between 1 and 2 displayed almost identical spectra except for two additional olefenic signals (H 5.22, 5.14), and four methyl doublet signals (H 0.94, 0.88, 0.82, 0.79) that replaced the methyl doublet and isopropyl doublet. The two olefenic protons H 5.22 and 5.14 (H-23 and H-22) had a COSY correlation with a large homonuclear coupling (J = 15.3 Hz), indicating the presence of a trans-double bond. The shift of the H3-18 and H3-21 methyls to more deshielded resonances (H 0.68 and H 0.94) in addition to a HMBC correlation from H

0.94 to C 135.5 (C-22) allowed the trans-double bond to be assigned at C-22 of the acyclic chain. Further HMBC correlations from the methyl resonance at H 0.88 to C 131.8, 42.5 and 32.7 (C-23, C-24 and C-25, respectively), as well as the methyl resonances at H 0.82 and 0.79 to C 42.5, 32.7 (C-24 and C-25, respectively), allowed the assignment of the extra C-28 methyl to the acyclic chain.

COSY HMBC Figure 4-7: Key COSY and HMBC correlations of the side chain of 2. ROESY correlations indicated the same configuration as 1, however no configuration was assigned for the additional C-24 stereogenic centre due to a lack of definitive ROESY correlations required for its assignment. Previously, this methyl was reported to be in the S configuration by analogy with previously reported NMR shifts.4 However, on analysis of these previously reported shifts,5 we believe the difference in methyl proton resonances between the S and R configurations (H-28S - H-28R = 0.001 ppm) is not great enough to distinguish the configuration at this position. Due to the low yield of this compound, we suggest that the only way to correctly assign the configuration of C- 24 is through synthesis of the 24S and 24R enantiomers for a complete comparison of spectroscopic data. Therefore, Dysidea-sterol B-11-acetate was assigned as (22E)- 2α,3β,5α,6β,9α,19-hexahydroxy-24-methylcholest-7,22-diene-11α-acetate.

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Structure elucidation of Dysidea-sterol A-11,19-diacetate (3) Dysidea-sterol A-11,19-diacetate (3) could only be isolated as a mixture with its C-22 olefin derivative 4 (2:1 molar ratio). A weak sodiated molecule [M+Na]+ was observed at m/z 589.3340 in the (+) HRESIMS, consistent with a molecular formula of C31H50O9. The structure of 3 was elucidated from NMR analysis and by analogy with 1.

21 22 18 24 AcO H 26 AcO 17 19 11OH 13 HO 1 9 15 H HO 3 5 7 OH OH 3 Figure 4-8: Structure of Dysidea-sterol A-11,19-diacetate (3).

Table 4-3: NMR data for Dysidea-sterol A-11,19-diacetate (3) in DMSO-d6.

Pos. δC, Type δH (J in Hz) COSY HMBC ROESY 1 α 30.9, CH2 1.91, m 1β, 2 2, 10, 19 3 β 2.12, dd (13.4, 4.2) 1α, 2 2, 3, 5, 10 2, 19 2 β 71.4, CH 3.10, ddd (12.0, 9.2, 4.3) 1α, 1β, 3 3 1β, 4β, 19 3 α 69.9, CH 3.51, m 2, 4α, 4β 1α, 4α 4 α 38.7, CH2 1.58, dd (14.2, 5.4) 3, 4β 2, 3, 5, 6, 10 3 β 1.92, dd (14.1, 11.7) 3, 4α 3, 6, 10 19 5 76.0, C 6 α 70.5, CH 3.52, dd (5.7, 2.3) 7, 14 5, 7, 8, 10 7 7 122.7, CH 5.26, dd (5.8, 2.3) 6, 14 5, 9, 14 6, 15β, 15α 8 139.3, C 9 74.9, C 10 46.4, C 11 β 73.6, CH 4.70, dd (11.5, 4.7) 12α, 12β 11-OAc 12β, 18, 19b 12 α 40.2, CH2 1.53, t (11.6) 11, 12β 11, 13, 17, 18 14, 17 β 2.08, dd (11.6, 4.8) 11, 12α 9, 11, 14 11, 18, 21 13 41.9, C 14 α 50.0, CH 2.44, ddt (12.4, 6.6, 2.3) 6, 7, 15β, 15α 7, 8, 12, 13, 15 12α, 15α, 16α, 17 15 α 22.4, CH2 1.47, dt (13.2, 6.6) 14, 15β, 16β 13 7, 14,16β β 1.35, m 14, 15α, 16β 14 7, 16β, 18 16 α 27.4, CH2 1.29, m 16β 17, 18, 20 18, 14 β 1.86, ddd (12.7, 6.4, 3.2) 15α, 15β, 16α 15α, 15β 17 α 55.3, CH 1.24, m 20, 16β 13, 16, 18, 20 12α, 14 18 12.0, CH3 0.57, s 12, 13, 14, 17 11, 12β, 15α, 16α, 20 19 a 64.4, CH2 4.25, d (13.7) 19b 5, 9, 10 2, 4β b 3.77, d (13.7) 19a 1, 5, 9, 10 1β, 11 20 35.6, CH 1.33, m 17, 21, 22 18 21 18.5, CH3 0.84, d (6.7) 20 17, 20, 22 12β 22 35.4, CH2 1.30, m 20, 23 0.96, m 20, 23 23 22.3, CH2 1.32, m 20, 23 1.13, m 20, 23 24 38.9, CH2 1.11, m 22, 24 1.07, m 22, 24 25 27.5, CH 1.48, m 22, 24 26 22.7, CH3 0.83, d (6.6) 25 24, 25, 27 27 22.7, CH3 0.83, d (6.6) 25 24, 25, 26 11-OAc 169.4, C 21.8, CH3 2.03, s 11-OAc 19-OAc 170.4, C 20.5, CH3 1.90, s 19-OAc 117

Comparison of the NMR and MS data between 1 and 3 indicated that the two molecules had similar structural features. The analysis of the 1H NMR and HSQC data (Table 4-3) indicated that the only additional signal was an acetate methyl (H 1.90). However, it also indicated significant differences for the chemical shifts associated with H-11 (H

13 4.70), H2-19 (H 4.25/3.77), H-2 (H 3.10), H-1β (H 2.12), and H3-18 (H 0.57). The C

NMR confirmed the presence of an additional acetate (C 170.4, 20.6) in the steroid.

Figure 4-9: 1H NMR spectrum of a mixture of Dysidea-sterol A-11,19-diacetate (3) and Dysidea-sterol B-11,19-

diacetate (4) in DMSO-d6.

A HMBC correlation from H 4.25 (H-19a) to the carbonyl at C 170.4 (19-OAc), in addition to the further deshielding of the H2-19 resonances, allowed the acetate to be assigned on C-19 of the steroid. The other changes in the 1H NMR resonances described above were also in agreement with the assignment of the acetate. ROSEY correlations allowed the assignment of relative configuration of all eleven stereogenic centres to be in agreement with the configuration of 1. Dysidea-sterol A-11,19-diacetate (3) was therefore assigned as 2α,3β,5α,6β,9α-pentahydroxycholest-7-ene-11α,19-diacetate.

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Figure 4-10: Configuration of the diacetate Dysidea-sterol A-11,19-diacetate (3).

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Structure elucidation of Dysidea-sterol B-11,19-diacetate (4) Dysidea 5α-sterol B-11,19-diacetate (4) could only be isolated as a mixture with 3 (1:2 molar ratio). A weak sodiated molecule [M+Na]+ was observed at m/z 601.3341 in the

(+) HRESIMS, consistent with a molecular formula of C32H50O9. The structure of 4 was elucidated from NMR analysis and by analogy with 2.

21 28 22 18 24 AcO H 26 AcO 17 19 11OH 13 HO 1 9 15 H HO 3 5 7 OH OH 4 Figure 4-11: Structure of Dysidea-sterol B-11,19-diacetate (4).

Table 4-4: NMR data for Dysidea-sterol B-11,19-diacetate (4) in DMSO-d6.

Pos. δC, Type δH (J in Hz) COSY HMBC ROESY 1 α 30.9, CH2 1.91, m 1β, 2 2, 10, 19 3 β 2.12, dd (13.4, 4.2) 1α, 2 2, 3, 5, 10 2, 19 2 β 71.4, CH 3.10, ddd (12.0, 9.2, 4.3) 1α, 1β, 3 3 1β, 4β, 19 3 α 69.9, CH 3.51, m 2, 4α, 4β 1α, 4α 4 α 38.7, CH2 1.58, dd (14.2, 5.4) 3, 4β 2, 3, 5, 6, 10 3 β 1.92, dd (14.1, 11.7) 3, 4α 3, 6, 10 19 5 76.0, C 6 α 70.5, CH 3.52, dd (5.7, 2.3) 7, 14 5, 7, 8, 10 7 7 122.7, CH 5.26, dd (5.8, 2.3) 6, 14 5, 9, 14 6, 15β, 15α 8 139.3, C 9 74.9, C 10 46.4, C 11 β 73.6, CH 4.70, dd (11.5, 4.7) 12α, 12β 11-OAc 12β, 18, 19b 12 α 40.2, CH2 1.50, t (11.6) 11, 12β 11, 13, 17, 18 14, 17 β 2.12, dd (11.6, 4.8) 11, 12α 9, 11, 14 11, 18, 21 13 41.9, C 14 α 50.0, CH 2.44, ddt (12.4, 6.6, 2.3) 6, 7, 15β, 15α 7, 8, 12, 13, 15 12α, 15α, 16α, 17 15 α 22.4, CH2 1.47, dt (13.2, 6.6) 14, 15β, 16β 13 7, 14,16β β 1.35, m 14, 15α, 16β 14 7, 16β, 18 16 α 27.5, CH2 1.29, m 16β 17, 18, 20 18, 14 β 1.69, ddd (12.7, 6.4, 3.2) 15α, 15β, 16α 15α, 15β 17 α 54.9, CH 1.30, m 20, 16β 13, 16, 18, 20 12α, 14 18 12.0, CH3 0.58, s 12, 13, 14, 17 11, 12β, 15α, 16α, 20 19 a 64.4, CH2 4.25, d (13.7) 19b 5, 9, 10 2, 4β b 3.77, d (13.7) 19a 1, 5, 9, 10 1β, 11 20 39.7, CH 1.97, m 17, 21, 22 18 21 20.5, CH3 0.92, d (6.7) 20 17, 20, 22 12β 22 136.1, CH 5.12, dd (15.3, 8.5) 20, 23 23 132.3, CH 5.19, dd (15.3, 7.7) 22, 24 24 41.9, CH 1.82, h 23, 25, 28 25 32.5, CH 1.43, m 24, 26, 27 26 19.5, CH3 0.78, d (6.8) 25 24, 25, 27 27 20.1, CH3 0.80, d (6.6) 25 24, 25, 26 28 18.0, CH3 0.87, d (6.9) 24 23, 24, 25 11-OAc 169.5, C 21.8, CH3 2.02, s 11-OAc 19-OAc 170.4, C 20.5, CH3 1.90, s 19-OAc 120

Comparison of the NMR and MS data between 2 and 4 suggested that, like 3, 4 is a diacetate form of 2. The analysis of the 1H NMR and 13C NMR data (Table 4-4) supported this conclusion with the only additional signals associated with an acetyl group (H 1.90, C 170.4, 20.6). ROSEY correlations allowed eleven of the twelve stereogenic centres to be assigned in the same configuration as 1. However, similar to 2, a lack of definitive ROESY correlations did not allow the C-24 configuration to be assigned. Dysidea-sterol B-11,19-diacetate (4) was therefore assigned as (22E)-24- methyl-2α,3β,5α,6β,9α-pentahydroxycholest-7,22-ene-11α,19-diacetate.

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Biological evaluation of the Dysidea-sterols The chemistry of Dysidea species has been extensively studied. The majority of natural products discovered from this genus are merosesquiterpenes,6 brominated diphenyl ethers,7 and sterol derivatives.8 The merosesquiterpenes have been found to have a broad range of bioactivities, including anti-microbial,9 anti-cancer,10 anti-viral,11 anti- inflammatory12 and anti-acetylcholinesterase13 activities. The brominated diphenyl ethers have been shown to exhibit antimicrobial activity14 and the sterol derivatives have been shown to display moderate anti-microbial15 and anti-cancer activity.8

Even though Dysidea natural products have been extensively evaluated, they have not yet been screened for anti-prion activity and only three compounds have been screened in assays related to neurodegenerative diseases. These compounds are the merosesquiterpenes, avarol (5),13 dysideamine (6) and bolinaquinone (7),16 and they have been reported to exhibit moderate inhibition of acetylcholinesterase. This activity may be useful for the treatment of neurodegenerative diseases.

O HO HO

OH H H R O

5 6: R = NH2 7: R = OCH3 Figure 4-12: Merosesquiterpenes isolated from the Dysidea genus that inhibit acetylcholinesterase.

The Dysidea-sterols (1-4) were first isolated in 1988 from the sponge, Dysidea etheria.4 These sterols were also reported and analysed as mixtures as no separation between the acyclic chain analogues was achievable. A defining feature of the poly-oxygenated sterols isolated from Dysidea species is C-19 oxygenation and this is uncommon in steroids isolated from other organisms. Four other related C-19 oxygenated poly- oxygenated sterol structures have been isolated from Dysidea species (8-11).17-20

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H H HO HO O O H HO H H HO HO OH R OR 8 OH 10 R

O H HO H HO OH OH HO OH H H HO HO OH OH 9 OH 11 Figure 4-13: The oxygenated C-19 sterols isolated from the Dysidea genus: 9/11-epoxy sterols (8),17 11/19-ether sterols (9),18 herbasterols (10)19 and 5β-dysidea-sterols (11).20

The Dysidea-sterol acetate and diacetate mixtures (1&2 and 3&4, respectively) were evaluated for their anti-prion activity using the yeast-based anti-prion bioassay described in Chapter One. This assay makes use of the [PSI+] and [URE3] prions to evaluate the ability of compounds to cure yeast prion infection. This in vivo yeast-based assay is useful as it allows curing to be tested following interference with any one of a number of different cellular prion mechanisms in addition to direct physical interaction of the compound with the prion. The sterol acetate mixture (1&2) and diacetate mixture + (3&4) were found to cure the [PSI ] prion with EC50s of 29.4 μM and 30.1 μM, respectively (Table 4-5). These poly-oxygenated sterols have comparative potency to that of other highly potent anti-prion compounds such as guanabenz.21 These Dysidea- sterols, however, also exhibit anti-fungal activity at higher doses. At concentrations greater than 50 μM yeast growth was so strongly inhibited (<70% inhibition of growth) that the colour readout used to quantify anti-prion activity could not be detected. Therefore, the activity of these sterols against the [URE3] prion could not be assessed. Interestingly, however, the crude extract they were isolated from was found to display anti-prion activity against the [URE3] prion and in the bioassay-guided isolation only those fractions that contained the poly-oxygenated sterols were observed to have anti- prion activity.

Table 4-5: Anti-prion activity of the poly-oxygenated Dysidea-sterols. Yeast [PSI+] prion Yeast [URE3] prion Compounds activity activity IC50 ± SE (µM) Yes/No Dysidea-sterol acetate (1 & 2) 29.4 ± 7.8 n.d.* Dysidea-sterol diacetate (3 & 4) 30.1 ± 4.2 n.d.* Guanabenz 27.4 ± 5.2 Yes *anti-prion activity could not be assayed due to toxicity

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The Dysidea-sterols (4-7) described in this chapter are the first compounds from a Dysidea species that have been reported to possess anti-prion activity. Interestingly, the didemnisterols described in Chapter Three are also poly-oxygenated sterols with anti- prion activity. The Dysidea-sterols isolated lack a sulfate in the side chain, contain an extra degree of unsaturation and have a different oxygenation pattern. With the discovery of this second set of poly-oxygenated sterols from the marine environment with anti-prion activity we suggest the examination of a wider range of poly-oxygenated sterols for their activity against prions and neurodegenerative disorders.

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EXPERIMENTAL SECTION General Experimental Procedures NMR spectra were recorded at 25 °C on a Bruker BioSpin GmbH 800 MHz spectrometer with a triple (TCl) resonance 5 mm cryoprobe. The DMSO-d6 peak was referenced in the NMR to H 2.50 and C 39.52. High resolution electrospray ionisation TOF MS data were acquired using an Agilent 6530-accurate mass Q-TOF LC/MS mass spectrometer equipped with a 1200 Series autosampler and 1290 Infinity HPLC. Altech

Davisil 35-76 m 60 Å C18 silica was used to adsorb samples prior to HPLC separation. A Merck Hitachi L7100 pump and a L7455 PDA detector were used for HPLC. HPLC columns used for separation were a Thermo Betasil C18 5 m, 100 Å, 150 mm x 21.2 mm column and a YMC diol bonded Silica 5 m, 100 Å, 150 mm x 20.0 mm column.

The solvents used for HPLC and MS analysis were HPLC grade and the H2O was Millipore Milli-Q PF-filtered. Trifluoroacetic acid (TFA) was spectroscopy grade from Alfa Aesar. Guandine hydrochloride (GuHCl) and guanabenz were analytical grade from Sigma. The S. cerevisiae STRg6 strain was used to screen for anti-prion activity against the [PSI+] prion (MATa ade1-14 trp1-289 his3Δ200 ura3-52 leu2-3,112 erg6::TRP1 [PSI+]). The S. cerevisiae SB34 strain was used to screen for anti-prion activity against the [URE3] prion (MATa ade2-1 trp1-1 leu2-3,112 his3-11,15 ura2::HIS3 dal5::ADE2 [URE3]). Starstedt sterile 96-well plates were used for screening.

Animal Material A sample of Dysidea sp. was collected on 21st June, 2011 using scuba in coastal waters off Coffs Harbour, NSW, Australia. The animal material was freeze-dried and stored at room temperature. A voucher specimen (ACENV0258) is located at Griffith University, Gold Coast, Queensland, Australia. The sponge was taxonomically identified by A.C.

Extraction and Isolation A freeze-dried sample of Dysidea sp. (8.62 g) was extracted exhaustively with MeOH to yield a crude extract (0.5893 g). The extract was adsorbed onto C18 silica gel in a 1:1 ratio and packed into a refillable HPLC cartridge (10 mm x 20 mm). The cartridge was connected in series to a Betasil C18 bonded silica HPLC column (21 mm x 150 mm).

The columns were eluted with a gradient from 100% H2O containing 0.1% TFA to 100% MeOH containing 0.1% TFA over 60 min at a flow rate of 9 mL/min. The column was further eluted with 100% MeOH containing 0.1% TFA for 10 min. A total

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of 70 fractions were collected at one min intervals. 0.15 mL aliquots (1.7% of each fraction) from every seven sequential fractions were combined to obtain ten fractions for anti-prion testing. Active fractions were analysed by 1H NMR and subsequently fraction 54 (2.31 mg) was dissolved in 10 μL of MeOH for further separation. Fraction 54 was separated by injecting the solution onto a diol bonded silica HPLC column (20 mm x 150 mm). The column was eluted with a gradient from 100% hexane to 100% DCM over 25 min, then to 10% MeOH/90% DCM over 25 min, and then to 100% MeOH over 25 min at a flow rate of 9 mL/min. The column was then further eluted with 100% MeOH for 10 min. A total of 85 fractions were collected at one min intervals. Fraction 52-54 contained the Dysidea-sterol diacetate mixture (4-5, 0.4 mg, 4.6 x 10-3 % dry wt) and fractions 55-58 contained the Dysidea-sterol acetate mixture (6-7, 0.4 mg, 4.6 x 10-3 % dry wt).

Dysidea-sterol A-11-acetate (1) and Dysidea-sterol B-11-acetate (2); clear amorphous solid; 1H NMR and 13C NMR see Table 4-1 and Table 4-2. (+) HRESIMS m/z [M + + + + Na] 547.3235 (calcd for C29H48O8Na 547.3247) and m/z [M + Na] 559.3238 (calcd + for C30H48O8Na 559.3247).

Dysidea-sterol A-11,19-diacetate (3) and Dysidea-sterol B-11,19-diacetate (4); clear amorphous solid; 1H NMR and 13C NMR see Table 4-3 and Table 4-4. (+) HRESIMS + + + m/z [M + Na] 589.3340 (calcd for C31H50O9Na 589.3353) and m/z [M + Na] 601.3341 + (calcd for C32H50O9Na 601.3353).

In Vivo Yeast-Based Anti-Prion Assay The anti-prion activity was determined by screening compounds using the yeast strains containing the [PSI+] or [URE3] prions. The STRg6 Saccharomyces cerevisiae strain was used for testing against the [PSI+] prion and the SB34 S. cerevisiae strain was used for testing against the [URE3] prion. Both strains have been genetically modified to introduce a mutation in an adenine biosynthesis biomarker gene, ADE1 or ADE2, respectively, which causes the yeast cultures to be white when the respective protein, Sup35p or Ure2p, is in its prion-infected form and red when the respective protein is in its normal uninfected form. Assays were performed in 96-well plates which have a working volume of 200 μL or in 384-well plates that have a working volume of 50 μL. Plates were incubated for 48 hrs at 24°C to allow for growth of the yeast and then for a further 48 hrs at 4°C for the red colour to develop. The red colour intensity was quantified by measuring fluorescence (ex. 544 nm/em. 620 nm) using a BMG Labtech

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FLUOstar Omega microplate fluorimeter. The percentage curing of the prion infection was calculated by comparison of the colour intensity of treated samples with that of the negative (uncured) and positive (cured) controls. The negative control was 2% w/v

DMSO (0% curing) and the positive control was 2 mM GuHCl (100% curing). EC50 values for activity against the [PSI+] prion were calculated from the dose-response curves for each compound.

REFERENCES (1) Prusiner, S. B. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 13363-13383. (2) Aguzzi, A.; Lakkaraju, A. K.; Frontzek, K. Annu. Rev. Pharmacol. Toxicol. 2018, 58, 331-351. (3) Jennings, L. K.; Ahmed, I.; Munn, A. L.; Carroll, A. R. Prion 2018, 12, 234-244. (4) West, R. R.; Cardellina, J. H. J. Org. Chem. 1988, 53, 2782-2787. (5) Rubinstein, I.; Goad, L. J.; Clague, A. D. H.; Mulheirn, L. J. Phytochemistry 1976, 15, 195-200. (6) Abdjul, D. B.; Yamazaki, H.; Takahashi, O.; Kirikoshi, R.; Ukai, K.; Namikoshi, M. J. Nat. Prod. 2016, 79, 1842-1847. (7) Fu, X.; Schmitz, F. J. J. Nat. Prod. 1996, 59, 1102-1103. (8) de Almeida Leone, P.; Redburn, J.; Hooper, J. N. A.; Quinn, R. J. J. Nat. Prod. 2000, 63, 694-697. (9) Seibert, G.; Raether, W.; Dogovic, N.; Gasic, M. J.; Zahn, R. K.; Muller, W. E. Zentralbl Bakteriol Mikrobiol Hyg A 1985, 260, 379-386. (10) Božić, T.; Novaković, I.; Gašić, M. J.; Juranić, Z.; Stanojković, T.; Tufegdžić, S.; Kljajić, Z.; Sladić, D. Eur. J. Med. Chem. 2010, 45, 923-929. (11) Sarin, P. S.; Sun, D.; Thornton, A.; Muller, W. E. J. Natl. Cancer Inst. 1987, 78, 663-666. (12) Ferrándiz, M. L.; Sanz, M.-J.; Bustos, G.; Payá, M.; Alcaraz, M.-J.; De Rosa, S. Eur. J. Pharmacol. 1994, 253, 75-82. (13) Pejin, B.; Iodice, C.; Tommonaro, G.; De Rosa, S. J. Nat. Prod. 2008, 71, 1850- 1853. (14) Handayani, D.; Edrada, R. A.; Proksch, P.; Wray, V.; Witte, L.; Van Soest, R. W. M.; Kunzmann, A.; Soedarsono J. Nat. Prod. 1997, 60, 1313-1316. (15) Lu, Y.; Zhao, M. Z. Naturforsch. B Chem. Sci. 2017, 72, 49-52. (16) Suna, H.; Arai, M.; Tsubotani, Y.; Hayashi, A.; Setiawan, A.; Kobayashi, M. Bioorganic Med. Chem. 2009, 17, 3968-3972. (17) Gunasekera, S. P.; Schmitz, F. J. J. Org. Chem. 1983, 48, 885-886. (18) Braekman, J. C.; Daloze, D.; Moussiaux, B.; Vandevyver, G.; Riccio, R. Bull. Soc. Chim. Belg. 1988, 97, 293-296. (19) Capon, R. J.; Faulkner, D. J. J. Org. Chem. 1985, 50, 4771-4773. (20) West, R. R.; Cardellina, J. H. J. Org. Chem. 1989, 54, 3234-3236.

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CHAPTER 5:

Screening natural products for anti-prion activity and evaluation of isolated anti- prion compounds for selection as lead chemical scaffolds

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ABSTRACT To date the majority of studies conducted to identify novel anti-prion compounds for therapeutic use have largely failed to identify any compounds with sufficient activity in vivo. This has primarily been due to the poor passage through the blood-brain barrier, and the toxic side effects of these compounds. In our recent study to identify new anti- prion compounds by screening natural product libraries, we reported the identification of 16 compounds with novel anti-prion activity. Subsequently, we evaluated these compounds for their potential as lead candidates for the design of new anti-prion therapeutic drugs. This evaluation led to the findings that: 1. a number of the natural products isolated in this study have physicochemical properties analogous to current CNS drugs, and 2. that they have low toxicity compared to previous anti-prion compounds. The results of an in vitro α-synuclein aggregation inhibitory assay led us to hypothesise that these natural products may have a direct mode of action to inhibit prion formations. We believe that further evaluation of these compounds and more extensive screening of natural extract and compound libraries for anti-prion activity would be highly beneficial.

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INTRODUCTION Chapters one to four have all described the isolation, structure elucidation, and anti- prion activity of marine natural products from four different invertebrate species. This study has been successful in identifying a number of potent anti-prion natural products. The anti-prion guided isolation led to the identification of nine new and seven known natural products with novel anti-prion activity. These compounds included three bromotyrosine alkaloids (1-3) from the sponge S. ianthelliformis, four butenolide (4-7) and two diphenylpropanone (8-9) metabolites from the ascidian P. procera, three sulfated poly-oxygenated sterols (10-12) from the ascidian Didemnum sp., and four acetylated poly-oxygenated sterols (13-16) from the sponge Dysidea sp.

R1 Br N R2 R1 O R Me2N O MeO O MeO OH OR O Br 2 1: R1 = R2 = Me Br Br

Br Br OH OMe N Br Br 2: R1 = H, R2 = OH OH Br 4: R = R = H O 1 2 8: R = H 5: R = Br, R = H HO Br 1 2 9: R = Br N O 6: R1 = H, R2 = Me 3: R1 = H, R2 = OMe 7: R1 = Br, R2 = Me

O Br

R R2 H AcO H R O H 1 OH HO H OH H HO OH HO H OH OH

10: R = OSO3H 13: R1 = H, R2 =

OSO H 11: R = 3 14: R1 = H, R2 =

12: R = OSO H 3 15: R1 = Ac, R2 =

16: R1 = Ac, R2 = Figure 5-1: The anti-prion compounds identified in the current bio-discovery effort.

Earlier, we reviewed the current literature on anti-prion therapeutic leads and concluded that there were two main complications that had led to the lack of an approved anti- prion drug. These were the efficiency with which the compounds permeate the blood- brain barrier (BBB) and their toxic side effects. In Chapter One, we reported that one of 130

the benefits of using an in vivo yeast-based bioassay is the ability to target the isolation to more drug-like molecules that can pass through the cell wall and the cell membrane. Additionally, to be scored as positive in this bioassay these compounds need to cure the yeast of the prion infection and restore normal biological function to the prion-infected protein.1 Therefore, the compounds isolated in this study may be a useful starting point for the development of novel anti-prion therapeutics. However, many of these compounds need further biological and physicochemical evaluation to identify lead structures.

This chapter describes the further evaluation of the isolated natural products to identify lead chemical scaffolds for the development of novel anti-prion therapeutics. The ligand efficiency of these compounds was calculated for a comprehensive comparison to previous anti-prion compounds. These natural products were evaluated for their physicochemical properties to predict the efficiency with which they penetrate the BBB. They were evaluated for their cytotoxicity against a human neuroblastoma cell line to evaluate their neurotoxicity. Finally, the compounds were screened for their ability to inhibit aggregation of α-synuclein in an in vitro cell-free assay. This provided insight into the anti-prion mode of action. This experimental data allowed the selection of promising molecular scaffolds as leads for future development.

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RESULTS AND DISCUSSION Comparison of ligand efficiency Unlike some more common diseases, there are no therapeutic or biological guidelines for lead compounds with anti-prion activity. Therefore, we used as a guide to create a benchmark for activity of lead anti-prion compounds in the yeast-based bio-assay, compounds that have previously been evaluated for suitability as anti-prion drugs. The compounds that we used as a guide included quinacrine (17), guanabenz (18), anle138b (19), and 6-aminophenanthridine (20). Quinacrine (17) is a drug that was previously used for the treatment of malaria. It has had clinical trials for the treatment of vCJD, however, has not progressed due to its limited effectiveness and significant toxicity at useful concentrations in vivo.2 Guanabenz (18) is currently used as an anti-hypertensive drug. It has been tested in animal models and while it displays potent inhibition of prions in vivo it has not progressed due to its severe toxicity.3 Anle138b (19) is a compound that targets the oligomerization of prions and other misfolded proteins. Promising results from in vitro cell-based and in vivo mouse models have led to clinical development of this compound for the treatment of α-synuclein aggregation and prion diseases.4 6-aminophenanthridine (20) is a potent anti-prion compound in yeast-based and mammalian cell-based assays. This compound, however, has not been evaluated further.5

N Cl HN H OMe N NH N 2 NH Cl N Cl 17 18

HN N O

O Br N NH2 19 20

Figure 5-2: Anti-prion compounds selected for comparison to anti-prion activity of the isolated natural products: quinacrine (17), guanabenz (18), anle138b (19), and 6-aminophenanthridine (20).

We compared the ligand efficiency (calculated using the method described by Hopkins et al. 2004)6 of the natural products identified in the current study to these known anti- prion compounds above (Table 5-1). The ligand efficiency is a measure of the efficiency of binding of a ligand to its therapeutic target.6 A high ligand efficiency indicates that a

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high proportion of the ligand is involve in binding, where as a low ligand efficiency indicates that fragments of the molecule are not associated with binding.7, 8 Ligand efficiency is typically used in drug discovery for the identification of lead compounds or molecular fragments, and for the comparison of potency among compounds with differing molecular sizes.8, 9 Hopkins et al. (2014) reported that acceptable values of ligand efficiency for drug candidates was >~0.3.8 In our study we found that guanabenz (18), 6-aminophenanthridine (20), and anle138b (19) had a high ligand efficiency (≥~0.3), and quinacrine had a lower ligand efficiency (<0.3). Based on the ligand efficiency of these previously identified anti-prion compounds we suggest that this previously set benchmark of >~0.3 is an ideal benchmark for anti-prion lead candidates.

The bromotyrosine derivatives (1-3), procerolides (4-7) and procerones (8-9) had ligand efficiency values between 0.22 and 0.28, the didemnisterols (10-12) had a ligand efficiency value of ~0.20, and the Dysidea-sterols (13-16) had ligand efficiency values of 0.16 to 0.17. Aplysamine-1 (1), procerolide A (4), procerone A (8), and procerone B (9) have the best ligand efficiencies (0.26-0.28) of the isolated anti-prion natural products. These ligand efficiencies are close to the 0.3 benchmark, thus making them ideal candidates for further development. However, these anti-prion natural products (1- 16) have ligand efficiencies considerably lower than the 0.3 benchmark, ideal for lead candidates. This indicates that a large proportion of the molecule is not involved in the ligand-target binding.10 Therefore, these anti-prion natural products may be useful, however, may need significant development before they could be considered as lead candidates. We suggest the use of synthetic studies to identify important molecular fragments for binding and the removal, or replacement, of unnecessary molecular fragments.

Physicochemical properties of anti-prion natural products While there are no guidelines for prion bioactivity, there are a number of good physicochemical guidelines to consider when assessing the drug-like properties of compounds. Our investigation of the physicochemical properties of the anti-prion natural products (1-16) are summarised in Table 5-1. Lipinski’s rules are a common set of physicochemical properties that are used to predict the oral bioavailability of compounds.11 The anti-prion natural products isolated typically have violations to Lipinski’s rules due to an increased logP and molecular weight. However, it is important to note that many natural products that are oral drugs, likewise do not

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Table 5-1. The physicochemical properties and ligand efficiency of the identified anti-prion natural products. [PSI+] Ligand Prion Curing Lipinski’s rule properties tPSA efficiency 2 Compounds EC50 (μM) MW ClogP HBA HBD Violations Å = 1.4(pEC50)/N aplysamine-1 (1) 102.2 408.2 3.94 3 0 0 15.7 0.279 aplysamine-2 (2) 4.2 650.2 6.11 7 2 1 83.4 0.228 purealidine-Q (3) 2.4 745.1 5.93 8 2 1 92.6 0.225 procerolide A (4) 22.7 549.0 5.22 5 2 2 76.0 0.260 procerolide B (5) 34.2 627.9 5.90 5 2 1 76.0 0.240 procerolide C (6) 64.6 563.0 5.28 5 1 2 65.0 0.225 procerolide D (7) 67.1 641.9 5.96 5 1 2 65.0 0.216 procerone A (8) 29.4 509.0 4.37 4 2 1 66.8 0.276 procerone B (9) 33.2 587.9 5.05 4 2 2 66.8 0.261 didemnisterol A (10) 12.7 516.7 4.64 7 4 1 124.3 0.196 didemnisterol B (11) 13.8 514.7 4.15 7 4 1 124.3 0.194 didemnisterol C (12) 9.8 528.7 4.55 7 4 1 124.3 0.195 Dysidea-sterol 11-acetate (13/14) 29.4 524.7 3.38 8 6 2 147.7 0.171 Dysidea-sterol 11,19-diacetate (15/16) 30.1 566.7 4.25 9 5 1 153.8 0.158

Quinacrine (17) 215 400.0 6.13 4 1 1 36.9 0.183 Guanabenz (18) 26.0 231.1 2.98 4 4 0 74.3 0.456 anle138b (19) 39.2 343.2 5.31 4 1 1 42.9 0.294 6-aminophenanthridine (20) 36.2 194.2 2.88 2 2 0 38.4 0.416 MW: molecular weight, ClogP: calculated octanol-water partition coefficient, HBA: hydrogen bond acceptor, HBD: hydrogen bond donor, tPSA: topological polar surface area

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obey Lipinski’s rules and it is thought that evolution has led to natural products that contain molecular transporters to bypass cellular barriers.12

While Lipinski’s rules give a good indication of oral drug-like properties of a molecule, CNS-drug like properties are much more specific than the equivalent properties for oral drugs. However, more recently, due to the rapidly increasing prevalence of mental illness and neurodegenerative disease, pharmaceutical companies have set physicochemical guidelines for CNS drug leads.13 These guidelines include the evaluation of physicochemical properties of compounds for the prediction of BBB permeation.14 Permeation through the BBB is an essential consideration for any lead compounds and chemical scaffolds to be used for the treatment of neurodegenerative disorders. The BBB is a selectively-permeable membrane that separates the blood from the cerebrospinal fluid (CSF).15 Compounds that typically display good permeation through the BBB include those that are relatively lipophilic with a neutral charge and low-molecular weight. While highly lipophilic compounds (ClogP >5) will permeate better they will be pumped back out of the CSF by the ATP- binding cassette (ABC) transporter proteins within the CNS.16 As the BBB typically inhibits the transport of hydrophilic compounds, a physicochemical property of molecules that has been shown to be a good predictor of BBB permeation is the topological polar surface area (tPSA) of the molecule.17 The tPSA is a value that reflects the area of the molecular surface of a molecule that is associated with electronegative atoms, such as oxygen and nitrogen. Requirements for the development of CNS drugs include a desirable polar surface area between 40 - 90 angstroms squared (Å2) with a maximum limit of 20 - 120 Å2, a desirable ClogP of less than 3 with a maximum limit of 5, and a desirable molecular weight of less than 360 Da with a maximum limit of 500 Da.13, 18

To evaluate the BBB permeation potential we compared the tPSA and the ClogP values of the isolated natural products to those for a library of 120 marketed CNS drugs, and the 58 previously identified anti-prion leads described in the introduction. We found a large proportion of the current anti-prion leads that are reported to lack BBB permeability (experimentally), and did not have tPSA or ClogP values similar to the currently used CNS drugs that have been reported to pass the BBB. In contrast, the anti- prion natural products that we isolated in this study have tPSA or ClogP values that are similar to the CNS drugs (Fig. 5-3). This indicates that these compounds could be good chemical scaffolds for further development into therapeutics.

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Figure 5-3: 3D histogram of the ClogP vs. tPSA values for (a) 120 currently used CNS drugs, (b) the natural products isolated in this study (NP, 1-16), and (c) the other anti-prion compounds described in the introduction. While the natural products have tPSA or ClogP values that are closer to those of the CNS drugs, suggesting that the natural products could have better BBB permeation than a number of previous anti-prion leads, these molecules still have a number of physicochemical properties that would need to be changed for these compounds to be good anti-prion therapeutic leads.

The brominated tyrosine (1-3), butenolide (4-7), and propanone (8-9) compounds have good tPSA values (<90 Å2), but high ClogP values (~5-6 logP) and molecular weights (~500-750 Da). While this can increase lipophilicity, and thus BBB permeation, this would make these compounds more prone to being pumped out of the CSF by ABC transporter proteins. Our preliminary investigation of the structure-activity relationships of these compounds indicates that the aromatic rings are important for the observed anti-prion activity. However, the removal of bromine atoms would reduce both the molecular weight and ClogP values to levels similar to those of currently used CNS drugs. The physicochemical properties of 1-9 indicate that these compounds have a good potential for further synthetic development into anti-prion lead therapeutics.

The poly-oxygenated sterols (10-16) have good ClogP values (~4-5 logP) and molecular weights (~500 Da); however, they have high tPSA values (~120-150 Å2). While this may be an issue for their use as drugs, there is considerable precedence for natural steroid derivatives passing through the BBB.19 Therefore, the use of these physicochemical properties for steroid derivatives may not be as useful for prediction of BBB permeation. In addition, a number of studies have reported the in vivo neurological activity of various sulfated steroid derivatives that act on ligand-gated ion channels.20 While the sulfated poly-oxygenated sterols isolated in this study (10-12) differ by the position of the sulfate (on C-26 instead of C-3), these compounds would have a good likelihood of permeating the BBB due to their similar chemical structure. This indicates

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that the sterols 10-12 may be useful as anti-prion therapeutic leads. The additional poly- oxygenated sponge sterols (13-16), while similar to a number of neurologically active steroids have a much larger number of hydroxyl groups, which is likely to make them less permeable through the BBB. Synthetic studies to identify important hydroxy substitution patterns and the removal of non-essential hydroxyl groups may lead to the creation of useful lead structures. However, it may also be advantageous to evaluate the anti-prion activity of steroids found in extracts from a number of other organisms.

The natural products isolated in this study all successfully permeate the yeast cell wall and membrane. This together with our analysis of their physicochemical properties indicates that they might be good starting structures for further development. Nevertheless, these natural compounds may not permeate the BBB in their current form.

Assessment of the neurotoxicity of the anti-prion natural products The toxicity of a number of the current anti-prion leads has reduced their potential to be developed further into drugs. Additionally, of lead compounds that are evaluated clinically, neurotoxicity is the main reason for their withdrawal from clinical trials.21 Hence, the ability of the compounds to efficiently cure prion infections with limited cytotoxicity is an important criterion for the selection of lead compounds. Therefore, the anti-prion natural products were evaluated for their preliminary neurotoxicity in a cell viability assay using the human SHSY-5Y neuroblastoma cell line. These experiments were conducted by Fleur McLeary in Assoc. Prof. Shailendra Anoopkumar-Dukie’s laboratory at the School of Pharmacy and Pharmacology, Griffith University.

Figure 5-4. The cell viability of SHSY-5Y neuroblastoma cells following treatment with the anti-prion natural products at three concentrations. Additionally, guanabenz (18) and quinacrine (19) were included for comparison. The three controls from left to right are media (No cells), DMSO vehicle control (with cells) and no treatment control (with cells). Shown are the bars representing mean + SEM bars for each treatment done in triplicate. Interestingly, the neurotoxicity of the anti-prion natural products displayed a similar trend to the yeast toxicity, with the exception of the sterol derivatives. This could be due

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to the use of erg6Δ mutant yeast strains, which have a reduced level of cellular steroids. As such, the insertion of these modified sterols could have a greater impact. Treatment with aplysamine-1 (1) at a concentration of 100 μM resulted in ~100% cell viability, and treatment with aplysamine-2 (2) and purealidine-Q (3) at 100 μM both resulted in toxicity (<10% cell viability), but treatment at 10 μM resulted in ~100% cell viability. Treatment with procerolides (4-7) and procerones (9-10) at 100 μM all resulted in ~100% cell viability. Didemnisterol A (10) was found to be toxic at 100 μM, but not at 10 μM (~100% cell viability). Treatment with the monoacetate poly-oxygenated sterol (13/14) from the sponge, Dysidea sp., at 100 μM resulted in a cell viability of ~76%, and treatment with the diacetate poly-oxygenated sterol (15/16) at 100 μM resulted in no toxicity (~100% cell viability). Additionally, it is interesting to note that the treatment of cells with a number of these compounds actually increased the cell viability compared to the controls. This could be due to some protective effects from the natural products.

The neurotoxicity of the anti-prion natural products identified in this study was compared to that of two anti-prion compounds that have not progressed clinically due to toxicity: guanabenz (18), which is reported to exhibit severe toxicity, and quinacrine (17), which is reported to exhibit toxicity at high doses.22, 23 Our analysis yielded similar results with ~25% cell viability for treatment with guanabenz at 1 μM, and 0% cell viability for treatment with quinacrine at 100 μM. This indicates that aplysamine-1 (1), the procerolides (4-7) and procerones (9-10), and the Dysidea-sterols (13-16) have significantly lower toxicity towards cultured neuroblastoma cells than guanabenz or quinacrine. The other natural products isolated (2, 3, 10) showed comparable toxicity to cultured neuroblastoma cells as quinacrine. While the toxicity of these compounds is similar to that of quinacrine, these natural products have approximately two orders of magnitude greater anti-prion activity than quinacrine and with greater ligand efficiency. Therefore, these natural compounds may still be useful as anti-prion lead candidates.

The modest toxicity of these compounds for a human neuroblastoma cell line indicates that these natural products may be good lead compounds for further development. Aplysamine-1 (1), and the procerolides (4-7) and procerones (9-10) in particular showed no cytotoxicity even at the highest concentration tested and thus are significantly less toxic than previous anti-prion leads that have failed due to toxicity.

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Inhibition of α-synuclein amyloid formation by anti-prion natural products α-synuclein (α-syn) is a protein associated with the neurodegenerative disorders Parkinson’s disease and Multiple Systems Atrophy. This β-sheet-rich protein misfolds to form toxic oligomers and insoluble amyloid fibrils similar to those of the prion protein.24 Recent research has revealed that the misfolded α-syn is able to undergo self- replication, i.e. physical interaction between the misfolded protein and the native protein causes the misfolding.25, 26 This is similar to the infectious process characteristic of the human prion protein. Additionally, propagation of the misfolded form of α-syn requires similar chaperone proteins to prion propagation.27 Therefore, due to the links and similarities between α-syn amyloid formation and propagation with the human prion protein, the isolated anti-prion natural products were screened for inhibition of α-syn aggregation in vitro. This experimentation was performed by Mingming Xu in Prof. George Mellick’s laboratory at Griffith Research Institute for Drug Discovery. This enabled the classification of the compounds as direct inhibitors or indirect inhibitors of the aggregation of β-sheet-rich misfolded proteins.

Figure 5-5. A preliminary screen of the anti-prion natural products and known anti-prion compounds for inhibition of α-syn aggregation in vitro using ThT fluorescence. The controls are the 72 hour untreated negative control and the epigallocatechin gallate (EGCG) positive control. Shown are the bars representing the ThT fluorescence of samples that correlates to the α-syn aggregation. The preliminary screen of the compounds was done with one replicate at a 5:1 molar ratio with purified α-syn (5 moles of compound : 1 mole α-syn). The aggregation was evaluated by using the fluorescence of the amyloid-binding dye thioflavin-T (ThT). The fluorescence associated with the aggregation of α-syn was reduced to various extents when the assay was performed in the presence of the anti-prion natural products (Fig. 5- 5).

Treatment with aplysamine-1 (1) and purealidine-Q (3) both resulted in a ~50% reduction in the ThT fluorescence, indicating inhibition of α-syn aggregation. 139

Interestingly, aplysamine-2 (2) did not reduce the ThT fluorescence. We suggest that this is most likely an error due to the lack of replicates in light of the activity of 1 and 3 in the same assay.

Figure 5-6. A further screen of procerolides A-D (4-7) and procerone A (8) for inhibition of α-syn aggregation in vitro using ThT fluorescence. The controls are the 72 hour untreated negative control and the epigallocatechin gallate (EGCG) positive control. Shown are the bars representing mean of the ThT fluorescence + standard error bars for each treatment done in triplicate.

The procerolides (4-7) and procerone A (8) all reduced the ThT fluorescence by ~75- 85% (Fig. 5-5). Procerone B (9) displayed significantly weaker activity (Fig. 5-5), and this again was assumed an error in light of the activity of the related compounds. Due to the large reduction in ThT fluorescence, these compounds were evaluated further with a greater number of replicates. These further assays indicated a large reduction (80-85%) in the fluorescence compared to the negative control (Fig. 5-6). Additionally, the structure-activity relationships for the compounds in this assay were consistent with those observed in the yeast-based anti-prion assay. The compounds with a hydrogen bond donor moiety on C-3 had slightly greater activity than those without this moiety. This indicates that the original hypothesis of the hydrogen bond donor playing and important role in the protein binding is likely true. The agreement between the structure- activity relationships in the two assays supports the views that the compounds have a similar and direct mode of action for inhibition of prion propagation in vivo and α-syn aggregation in vitro.

The poly-oxygenated sterol compounds that were screened for inhibition of α-syn aggregation include didemnisterol A (10) and the Dysidea-sterol acetate mixture (13- 14). These compounds were evaluated further with a greater number of replicates, and were found to reduce the ThT fluorescence by ~70% compared to the negative control (Fig. 5-7). Interestingly, there was no statistically significant difference between the levels of inhibition exhibited by the two compounds, indicating that the differences in

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activity observed for these two compounds in the yeast-based anti-prion assay may be due to differences in the efficiency with which they are taken up by yeast cells or in the cellular target relevant to their anti-prion activity.

Figure 5-7. A further screen of the poly-oxygenated sterols: didemnisterol A (10) and Dysidea-sterol-11-acetate mixture (13/14) for inhibition of α-syn aggregation in vitro using ThT fluorescence. The controls are the 72 hour untreated negative control and the epigallocatechin gallate (EGCG) positive control. Shown are the bars representing mean of the ThT fluorescence + standard error bars for each treatment done in triplicate. In the Introduction, we classified the previously identified anti-prion therapeutic leads into three categories: compounds that directly inhibit the conversion or aggregation of PrP, compounds that effectively clear the infectious PrPSc from the cells, and compounds that inhibit cellular mechanisms important for prion propagation. The results of this assay allow us to hypothesise that the natural products isolated in this study (1-16) could directly inhibit the conversion or aggregation of yeast prions. The anti-prion activity in yeast may be attributable to an ability of the natural products to inhibit protein aggregation. The mechanism by which this protein aggregation occurs may be conserved between yeast prions and human α-syn. Further evidence to supports this hypothesis is the striking similarity of the structure-activity relationships observed for the same compounds in the in vitro α-syn aggregation assay, and the in vivo yeast- based anti-prion assay.

Conclusion and selection of lead compounds In conclusion, these natural products have activity comparable to previously identified potent anti-prion compounds, with low toxicity to human neuroblastoma cells. The physicochemical properties indicate the potential of a number of these natural products to pass through the BBB and to meet therapeutic guidelines for CNS drugs following further development to reduce their logP value and molecular weight. The ligand efficiency indicates that further studies to identify important molecular fragments of these compounds needs to be performed. We recommend that the compounds isolated in

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this study be further evaluated in terms of structure-activity relationships. This should facilitate the identification of the features of the molecule important for the anti-prion activity, and the physicochemical properties for better BBB permeation potential. In particular, the compounds that showed the best results and that should be selected as lead compounds to be followed up for future development, are aplysamine-1 (1), procerolide-A (4), procerone-A (8), and didemnisterol-A (10). These compounds all displayed low toxicity and better BBB permeation potential than a number of previous lead compounds. Additionally, as these compounds directly inhibit the aggregation of α- syn, it would also be important to examine their ability to inhibit the aggregation of misfolded proteins associated with a number of other neurodegenerative diseases.

There are a number of other reported bromotyrosine, butenolide, and poly-oxygenated sterol compounds that are closely related in structure to those isolated in this study. We recommend testing these compounds for anti-prion activity to identify other anti-prion natural products. Based on the data in this chapter we suggest that the screening of more extracts derived from marine organisms would likely lead to more anti-prion lead compounds. The marine environment may be a fruitful source of drug-like anti-prion compounds.

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EXPERIMENTAL SECTION General experimental procedures The purification of the natural products (1-16) is described in Chapters 1-4. All compounds were dissolved in DMSO at a stock concentration of 10 mM. The physicochemical properties were calculated using the standard commercial packages: the calculation ClogP was obtained using BioByte, the calculation of tPSA was done using the Ertl methodology through http://www.molinspiration.com/.18 Analytical grade compounds, guanidine hydrochloride (GuHCl), quinacrine hydrochloride, and guanabenz were purchased from Sigma (Castle Hill, NSW, Australia). Anle138b was provided by Dr. Santosh Rudrawar’s laboratory (Griffith University, QLD, Australia), and 6-aminophenanthridine was provided by the Prof. Marc Blondel’s laboratory (CNRS, France). DMEM (Dulbecco's Modified Eagle Medium) with high glucose and pyruvate media, and fetal bovine serum were from Life Technologies/Gibco. The S. cerevisiae STRg6 strain was used to screen for anti-prion activity against the [PSI+] prion (MATa ade1-14 trp1-289 his3Δ200 ura3-52 leu2-3,112 erg6::TRP1 [PSI+]). The S. cerevisiae SB34 strain was used to screen for anti-prion activity against the [URE3] prion (MATa ade2-1 trp1-1 leu2-3,112 his3-11,15 ura2::HIS3 dal5::ADE2 [URE3]). Greiner sterile 96-well plates were used for screening. A BMG Labtech FLUOstar Omega microplate fluorimeter was use to analyse the fluorescence and absorption for the prion assay. A BioTek Synergy 2 microplate reader was used to measure fluorescence in the ThT assays.

In Vivo Yeast-Based Anti-Prion Assay The anti-prion activity was determined using published methodology.1 In short, compounds were screened against Saccharomyces cerevisiae harbouring either the [PSI+] or [URE3] prion. [PSI+] is the prion form of the Sup35p that is important for translation termination in yeast. [URE3] is the prion form of the Ure2p that is important for controlling the expression of nitrogen metabolism genes in yeast. Cultures of S. cerevisiae are white when Sup35p and Ure2p are in their prion-infected form and red when they are in their normal uninfected form. Assays were performed in 96-well plates with a working volume of 200 μL. Plates were incubated for 48 hrs at 24°C to allow the yeast to grow and then for a further 48 hrs at 4°C for the colour to develop. The red colour intensity was quantified by measuring fluorescence (ex. 544 nm/em. 620 nm) on a microplate fluorimeter. The percentage curing of the prion was calculated by

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comparison of the colour intensity of experimental test samples with that of DMSO (0% curing) and 2 mM GuHCl (100% curing) controls.

Assaying the effect of compounds on neuroblastoma cell viability The cell viability assay was performed with neuroblastoma cells using a resazurin assay. Resazurin (blue) can be reduced irreversibly to resorufin (pink) in direct proportion to the rate of aerobic respiration of the cells. A low cell viability will result in a higher concentration of resazurin. A high cell viability will result in a build-up of resorufin through the reduction of resazurin by the viable cells. A fluorimeter can be used to quantitatively analyse the colour, and a standard curve can be used to convert the output to percentage cell viability.28 An SHSY-5Y neuroblastoma cell culture was grown with incubation at 37°C with 5% CO2 in DMEM with high glucose and pyruvate, supplemented with 10% fetal bovine serum for 24 hours. This homogenous cell culture was distributed into the wells of 96-well plates with 180 μL per well. To each well, 20 μL of a natural product that was prepared at three concentrations (1 mM, 100 μM and 10 μM) in DMEM media and 10% DMSO was added. The treated cells were further incubated at 37°C with 5% CO2 for 24 hours. After 24 hours of treatment, resazurin was added and the plates were incubated for a further 3 hours at 37°C with 5% CO2. The plates were then analysed with a fluorimeter (ex. 530 nm/em. 590 nm). The fluorescence of the treated wells was compared to that of the media only control (no cells), positive control (100 μM cycloheximide to make cells inviable), negative control (media only) and DMSO vehicle control (1 %v/v DMSO). Percentage cell viability was calculated by taking the vehicle control value as 100% cell viability, and the positive control value as 0% cell viability. Assays were conducted by Fleur McLeary in Assoc. Prof. Shailendra Anoopkumar-Dukie’s laboratory (Griffith University, Australia).

ThT fluorescence-based α-synuclein aggregation assay A stock solution (5 mM) of thioflavin T (ThT) was prepared in glycine-NaOH buffer (pH 8.0). Homogenous monomeric α-syn was made by following the commonly used protocol by Rahimi et al.29 The α-syn monomers (80 μM) were dissolved in an aggregation buffer (20 mM Tris-HCl, pH 7.4, 150 mM NaCl and 0.05 % NaN3), and incubated at 37°C with constant shaking (1000 rpm in a thermomixer) for 72 hours in the presence or absence of the compounds (400 μM). Following incubation α-syn samples (60 μL) were mixed with the ThT solution (240 μL, 50 μM). Equal volumes of the 300 μL solution were then distributed into the wells of a 96-well plate, and the plates were analysed with a fluorimeter (ex. 440 nm/em. 500 nm). The fluorescence 144

intensity of the samples was compared to that of the negative control to obtain the percentage inhibition of aggregation. These assays were conducted by Mingming Xu in Prof. George Mellick’s laboratory (Griffith University, Australia).

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(23) Nguyen, P. H.; Hammoud, H.; Halliez, S.; Pang, Y.; Evrard, J.; Schmitt, M.; Oumata, N.; Bourguignon, J. J.; Sanyal, S.; Beringue, V.; Blondel, M.; Bihel, F.; Voisset, C. ACS Chem. Neurosci. 2014, 5, 1075-1082. (24) Brundin, P.; Melki, R. J. Neurosci. 2017, 37, 9808-9818. (25) Prusiner, S. B.; Woerman, A. L.; Mordes, D. A.; Watts, J. C.; Rampersaud, R.; Berry, D. B.; Patel, S.; Oehler, A.; Lowe, J. K.; Kravitz, S. N.; Geschwind, D. H.; Glidden, D. V.; Halliday, G. M.; Middleton, L. T.; Gentleman, S. M.; Grinberg, L. T.; Giles, K. Proc. Natl. Acad. Sci. U. S. A. 2015, 112, e5308-e5317. (26) Woerman, A. L.; Watts, J. C.; Aoyagi, A.; Giles, K.; Middleton, L. T.; Prusiner, S. B. Cold Spring Harb. Perspect. Med. 2018, 8. (27) Reyes, J. F.; Olsson, T. T.; Lamberts, J. T.; Devine, M. J.; Kunath, T.; Brundin, P. Neurobiol. Dis. 2015, 77, 266-275. (28) O'Brien, J.; Wilson, I.; Orton, T.; Pognan, F. FEBS J. 2000, 267, 5421-5426. (29) Rahimi, F.; Maiti, P.; Bitan, G. JoVE 2009, e1071.

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CONCLUSIONS:

Discovery of novel anti-prion compounds by screening natural product libraries using a yeast based assay: a success

147

This natural products bio-discovery project had three aims:

1. to develop a new anti-prion assay with the capacity of screening large and chemically complex natural extract libraries,

2. to isolate those natural products from the active extracts that are responsible for the observed anti-prion activity, and

3. to evaluate and compare these natural products to previously identified anti-prion compounds.

The first aim was achieved with the development of a simple yeast-based anti-prion assay (Chapter 1). This assay is useful for screening as anti-prion activity can reflect interference with any one of a number of conserved biological (or cellular) pathways in prion protein switching, aggregation and propagation, some of which can only be assayed in vivo. The use of yeast prions allows for a cheap, high-throughput and quantitative colorimetric screen. Additionally, the use of an in vivo yeast-based screen aids in the identification of more drug-like molecules that can pass through a cell wall and cell membrane. We would recommend the use of this assay for the continued screening of more naturally derived extracts. We believe that the further screening of marine extracts, in particular, would be important and result in the discovery of a greater number of anti-prion compounds.

The second aim was achieved with the isolation of sixteen anti-prion compounds from four marine invertebrate species. These included a diverse range of compounds including three bromotyrosine alkaloids (Chapter 1), four butenolide and two propanone metabolities (Chapter 2), and seven poly-oxygenated sterols (Chapters 3-4). These compounds all showed potent anti-prion activity that was comparable or greater than the previously identified and potently active anti-prion compounds: guanabenz, 6- aminophenanthridine and quinacrine. This suggests that the marine environment is a rich source of anti-prion compounds. Future work should also focus on the screening and evaluation of a number of known natural products that are closely related in structure to those isolated in this study.

The third aim was achieved though the comparison of the anti-prion natural products isolated in this study to those isolated in previous studies (Chapter 5). To date, no curative treatment is available for prion diseases, and a large number of the anti-prion lead compounds have failed to display activity in vivo. This has been primarily due to 148

the poor passage of those lead compounds through the BBB and for their severe toxicity. Of the compounds that effectively permeate the BBB without toxicity, none are effective when administered at the later stages of prion diseases. A comparison of the physicochemical properties and the toxicity of our anti-prion natural products, previous anti-prion lead compounds and current CNS drugs led us to conclude that the compounds identified in this study are more drug-like than a number of previous lead compounds. We identify a number of the isolated natural products as especially promising lead-like compounds that with further development could increase our current understanding of prion diseases. We suggest future work be focused on the structure-activity relationships of the lead compounds identified in this study. Additionally, given the observed ability of some of the identified compounds to inhibit α-synuclein aggregation, these compounds should be further evaluated against a range of other neurodegenerative diseases caused by misfolded proteins.

We believe that this project has been successful and has laid the foundation for future natural product projects focusing on the isolation of anti-prion compounds. Earlier, we presented the hypothesis that organisms in the marine environment may produce natural products to target misfolded proteins with important biological functions. The success of this project, with the isolation of new anti-prion active compounds, supports this view and indicates that naturally-derived extracts may be a rich and fruitful source of novel anti-prion compounds.

149

APPENDIX

150

SUPPLEMENTARY DATA - CHAPTER 1:

PUBLISHED:

Retrieved from Jennings, L. K.; Ahmed, I.; Munn, A. L.; Carroll, A. R. Prion 2018, 12, 234-244. https://www.tandfonline.com/doi/suppl/10.1080/19336896.2018.1513315?scroll=top

Supplementary data: Yeast-Based Screening of Natural Product Extracts Results in the identification of Prion Inhibitors from a Marine Sponge

Laurence K Jenningsa, Ishtiaq Ahmedb, Alan L Munnb, Anthony R Carrolla, c* a School of Environment and Science, Environmental Futures Research Institute,

Griffith University (Gold Coast campus), Parklands Drive, Southport, QLD 4222,

AUSTRALIA. b School of Medical Science and Understanding Chronic Conditions Program, Menzies

Health Institute Queensland, Griffith University (Gold Coast campus), Parklands Drive,

Southport, QLD 4222, AUSTRALIA. c Griffith Institute for Drug Discovery, Griffith University (Brisbane Innovation Park),

Don Young Road, Nathan, QLD 4111, AUSTRALIA.

* [email protected]

151

Isolation Data:

Freeze-dried Invertebrate Material (20.15 g) a

Invertebrate Extract (0.7973 g) b

Mixture Aplysamine-1 Purealidine-Q Aplysamine-2 (Elution: 14-16 min, (Elution time: (Elution time: (Elution time: 16.13 mg) 17 min, 9.64 mg) 37 min, 5.77 mg) 39 min, 7.96 mg) c

verongiaquinol verongiaquinol- (Elution time: 62 ketal min, 5.5 mg (Elution time: 64 min, 6.2 mg a. Methanol extraction b. Gradient reverse phase HPLC, 100% H2O to 100% MeOH over 60 minutes, betasil 5μm C18 silica column (21.2x150 mm) c. Gradient normal phase HPLC, 100% hexane to 100% DCM over 25 minutes, 100% DCM to 10% MeOH/DCM over 25 minutes, 10% MeOH/DCM to 100% MeOH over 25 minutes, betasil 5μm C18 silica column (21.2x150 mm) Supplementary Figure-S1: Isolation diagram of the anti-prion compounds.

Aplysamine-1 (1)

Br NMe2

Me2N O Br A dark yellow amorphous solid; The (+)HRESIMS observed m/z 409.0366 [M+H]+ + indicating a molecular formula C15H24Br2N2O (calcd C15H24Br2N2O•H m/z 409.0313). 1 H NMR (d6 DMSO, 500MHz) δ= 7.65 (s, 2H), 3.99 (t, J = 5.8 Hz, 2H), 3.32 (t, J = 8.8 Hz, 2H), 3.25 (t, J = 8.0 Hz, 2H), 2.95 (t, J = 8.0 Hz, 2H), 2.82 (s, 6H, NMe), 2.78 (s, 6H, NMe), 2.18 (m, 2H). 13C NMR δ= 151.3, 137.3, 133.7 (2C), 117.9 (2C), 70.7, 57.3, 54.6, 42.8 (4C), 28.8, 25.3.

Aplysamine-2 (2)

OH OMe N H Br N Br O Me2N O Br A dark yellow amorphous solid; The (+)HRESIMS observed m/z 649.9649 [M+H]+ + indicating a molecular formula C23H28Br3N3O4 (calcd C23H28Br3N3O4•H m/z 1 649.9688). H NMR (d6 DMSO, 500MHz) δ= 11.88 (s, 1H, OH), 8.04 (t, J = 5.8 Hz, 1H, NH), 7.48 (s, 2H), 7.37 (d, J = 2.0 Hz, 1H), 7.12 (dd, J = 2.0, 8.4 Hz, 1H), 6.98 (d, J = 8.5 Hz, 1H), 3.96 (t, J = 5.8 Hz, 2H), 3.79 (s, 3H, OMe), 3.71 (s, 2H), 3.35 (t, J = 7.2 152

Hz, 2H), 3.25 (t, J = 7.8 Hz, 2H), 2.76 (s, 6H, N-Me), 2.73 (t, J = 7.0 Hz, 2H), 2.14 (m, 2H). 13C NMR δ= 163.3, 153.8, 151.8, 150.3, 139.2, 132.8 (2C), 132.7, 130.5, 129.0, 117.2 (2C), 113.0, 110.4, 70.1, 56.0, 54.1, 42.3 (2C), 39.4, 33.2, 27.5, 24.7.

Purealidine-Q (3)

HO Br N O H Br N OMe

O Br Me2N O Br A dark yellow amorphous solid; The (+)HRESIMS observed m/z 745.8682 [M+H]+ + indicating a molecular formula C23H27Br4N3O5 (calcd C23H27Br4N3O5•H m/z 1 745.8721). H NMR (d6 DMSO, 500MHz) δ= 8.61 (t, J = 5.6 Hz, 1H, NH), 7.48 (s, 2H), 6.56 (s, 1H), 6.38 (d, J = 8.0 Hz, 1H, OH), 3.96 (t, J = 5.6 Hz, 2H), 3.91 (d, J = 8.0 Hz, 1H), 3.65 (s, 3H, OMe), 3.61 (d, J = 18.0 Hz, 1H), 3.37 (t, J = 7.1 Hz, 2H), 3.33 (t, J = 8.0 Hz, 2H), 3.18 (d, J = 18.3 Hz, 1H), 2.81 (s, 6H, N-Me), 2.76 (t, J = 6.9 Hz, 2H), 2.17 (m, 2H). 13C NMR δ= 159.5, 154.9, 150.6, 147.8, 139.7, 132.6 (2C), 130.8, 121.4, 117.7 (2C), 113.6, 91.0, 73.1, 69.8, 59.2, 53.9, 41.9 (2C), 39.3 (2C), 32.8, 24.4.

3,5-dibromoverongiaquinol (4)

OH Br NH2 O O Br A pale yellow amorphous solid; The (-)HRESIMS observed m/z 437.8642 [M+TFA-H]- - indicating a molecular formula C8H7Br2NO3 (calcd C8H7Br2NO3•CF3COO m/z 1 437.8623). H NMR (d6 DMSO, 500MHz) δ= 7.59 (s, 2H), 7.46 (bs, 1H, NH), 6.99 (bs, 1H, NH), 6.43 (bs, 1H, OH), 2.59 (s, 2H). 13C NMR δ= 172.5, 169.4, 153.4 (2C), 119.1 (2C), 71.9, 44.7.

3,5-dibromoverongiaquinol dimethyl ketal (5)

OH Br NH2 MeO O MeO Br A pale yellow amorphous solid; The (-)HRESIMS observed m/z 483.9064 [M+TFA-H]- - indicating a molecular formula C10H13Br2NO4 (calcd C10H13Br2NO4•CF3COO m/z 1 483.9041). H NMR (d6 DMSO, 500MHz) δ= 7.45 (bs, 1H, NH), 7.01 (bs, 1H, NH), 6.81 (s, 2H), 6.07 (bs, 1H, OH), 3.04 (s, 3H, OMe), 2.95 (s, 3H, OMe), 2.41 (s, 2H). 13C NMR δ= 170.1, 142.1 (2C), 120.5 (2C), 96.7, 70.6, 50.2 (2C), 46.6.

153

Supplementary Figure-S2: Dilutions of the 100% [PSI+] culture, 100% [psi-] culture and 50% [PSI+]/50% [psi-] culture were made. The absorbance of the samples was measured at 540nm to calculate the colour intensity and 600nm to calculate cell density. Absorbance at 540nm was graphed with absorbance at 600nm. The lower linear line is the dilutions of the 100% [PSI+] culture (blue). The upper linear line is the dilutions of the 100% [psi-] culture (red). The middle linear line is the dilutions of the 50% [PSI+]/50% [psi-] culture (purple). This was used confirm correction of colour intensity using the cell density.

Supplementary Figure-S3: Plot of the colour intensity of screening assays for all marine invertebrates vs. the optical density at 600nm as a measure of cell density. The lower linear line is 0% curing with the negative control (blue). The upper linear line is 100% curing with the positive control (red). The red line is the mean of the samples screened and the red dotted line indicates two standard deviations from the mean.

Supplementary Figure-S4: Micro-titre wells (a) and the dose response curves (b) of bioactive extracts that were confirmed active and were analysed further. 154

Supplementary Figure-S5: Dose response of compounds 1-3 with differing concentrations of GuHCl; (a) 0.5 mM GuHCl; (b) 0.25 mM GuHCl; (c) 0 mM GuHCl. This displays a clear synergistic effect between the three antiprion compounds described (1-3) and GuHCl.

Supplementary Table-S1: Activity data for each marine invertebrate extract screened.

ACENV No. Invertebrate STRg6 [PSI+] Confirmation SB34 [URE3] activity activity ACENV0028 Ascidian -5 ACENV0029 Sponge -11 ACENV0030 Sponge 12 ACENV0031 Sponge Toxic Inactive ACENV0032 Sponge 19 ACENV0033 Sponge Toxic Inactive ACENV0034 Ascidian Toxic Inactive ACENV0036 Sponge 31 ACENV0039 Ascidian 28 ACENV0040 Ascidian 9 ACENV0041 Ascidian 22 ACENV0043 Sponge 3 ACENV0045 Ascidian 12 ACENV0046 Sponge 15 ACENV0047 Sponge 19 ACENV0048 Sponge 8 ACENV0049 Sponge -2 ACENV0050 Sponge -1 ACENV0051 Sponge 5 ACENV0053 Sponge 12 ACENV0054 Sponge 20 ACENV0055 Sponge 9 ACENV0056 Sponge 11 ACENV0057 Sponge 21 ACENV0060 Sponge 14 ACENV0061 Sponge -5 155

ACENV0062 Sponge Toxic Inactive ACENV0063 Sponge -18 ACENV0064 Sponge -4 ACENV0065 Sponge 48 False positive ACENV0066 Ascidian 4 ACENV0067 Sponge 20 ACENV0068 Sponge 46 False positive ACENV0069 Sponge 13 ACENV0070 Sponge 18 ACENV0071 Ascidian 4 ACENV0072 Ascidian 39 ACENV0073 Soft Coral 8 ACENV0074 Ascidian 22 ACENV0076 Ascidian 17 ACENV0077 Ascidian 24 ACENV0078 Mollusc 19 ACENV0079 Ascidian 12 ACENV0080 Sponge 61 False positive ACENV0081 Sponge 16 ACENV0082 Sponge 18 ACENV0083 Sponge -12 ACENV0085 Sponge 7 ACENV0086 Ascidian 18 ACENV0088 Sponge 14 ACENV0089 Sponge 25 ACENV0090 Sponge 9 ACENV0091 Sponge 15 ACENV0092 Sponge -1 ACENV0093 Sponge 18 ACENV0094 Sponge 19 ACENV0095 Sponge 10 ACENV0096 Sponge 82 False positive ACENV0097 Sponge 16 ACENV0098 Sponge 16 ACENV0099 Sponge Toxic Inactive ACENV0100 Sponge 24 ACENV0101 Sponge Toxic Inactive ACENV0102 Sponge 24 ACENV0103 Ascidian 18 ACENV0104 Ascidian 16 ACENV0105 Ascidian 15 ACENV0106 Ascidian 27 ACENV0107 Ascidian 8 ACENV0108 Ascidian 26 ACENV0109 Ascidian 32 ACENV0111 Ascidian 34 ACENV0112 Ascidian 12 ACENV0113 Ascidian 19 156

ACENV0114 Ascidian 11 ACENV0115 Ascidian 17 ACENV0116 Ascidian 65 False positive ACENV0117 Ascidian 0 ACENV0119 Ascidian 20 ACENV0120 Ascidian 19 ACENV0121 Ascidian 10 ACENV0122 Ascidian 9 ACENV0124 Ascidian 30 ACENV0125 Ascidian 8 ACENV0127 Ascidian Toxic Inactive ACENV0128 Sponge 16 ACENV0129 Sponge 83 Active Active ACENV0130 Sponge 26 ACENV0131 Sponge -2 ACENV0132 Sponge 53 Active Inactive ACENV0133 Sponge 40 ACENV0134 Sponge 99 Active Active ACENV0135 Sponge 1 ACENV0136 Sponge 10 ACENV0137 Sponge 15 ACENV0138 Sponge 11 ACENV0139 Soft Coral 2 ACENV0140 Soft Coral 10 ACENV0141 Sponge 25 ACENV0142 Ascidian 16 ACENV0143 Ascidian 9 ACENV0144 Sponge 28 ACENV0145 Ascidian -3 ACENV0147 Sponge 26 ACENV0148 Ascidian 5 ACENV0149 Ascidian 31 ACENV0150 Ascidian 5 ACENV0151 Ascidian 17 ACENV0152 Mollusc 46 False positive ACENV0153 Sponge 24 ACENV0154 Ascidian 3 ACENV0155 Ascidian 9 ACENV0157 Sponge 16 ACENV0158 Sponge 15 ACENV0159 Sponge 11 ACENV0160 Sponge 17 ACENV0161 crinoid 64 False positive ACENV0162 Algae Toxic Inactive ACENV0163 Ascidian Toxic Inactive ACENV0166 Sponge Toxic Inactive ACENV0168 Sponge 20 ACENV0169 Sponge 22 157

ACENV0170 Sponge 31 ACENV0171 Sponge 42 Active Inactive ACENV0172 Sponge 10 ACENV0173 Sponge 18 ACENV0174 Sponge 18 ACENV0175 Sponge 17 ACENV0176 Sponge 11 ACENV0177 Sponge 15 ACENV0178 Sponge 20 ACENV0179 Ascidian 7 ACENV0180 Ascidian 12 ACENV0182 Ascidian 8 ACENV0183 Ascidian 34 ACENV0184 Soft Coral 29 ACENV0185 Soft Coral 1 ACENV0186 Zoanthid 20 ACENV0187 Ascidian 14 ACENV0188 Ascidian 24 ACENV0190 Ascidian 25 ACENV0193 Ascidian 3 ACENV0195 Sponge 26 ACENV0196 Ascidian 18 ACENV0197 Sponge 21 ACENV0198 Sponge 20 ACENV0199 Sponge 21 ACENV0200 Sponge 29 ACENV0201 Sponge 24 ACENV0202 Sponge 27 ACENV0203 Sponge 30 ACENV0204 Ascidian 17 ACENV0205 Sponge 21 ACENV0207 Sponge 15 ACENV0208 Sponge 21 ACENV0212 Sponge 21 ACENV0213 Sponge 34 ACENV0215 Sponge 25 ACENV0216 Ascidian 3 ACENV0217 Sponge 21 ACENV0218 Sponge 11 ACENV0219 Sponge 37 ACENV0220 Sponge 26 ACENV0221 Sponge 45 False positive ACENV0222 Sponge 10 ACENV0223 Sponge 21 ACENV0224 Sponge -1 ACENV0225 Sponge 1 ACENV0227 Ascidian 13 ACENV0230 Sponge 27 158

ACENV0231 Ascidian 18 ACENV0232 Ascidian 9 ACENV0233 Ascidian 11 ACENV0234 Sponge 20 ACENV0235 Ascidian 37 ACENV0236 Sponge 23 ACENV0237 Ascidian 7 ACENV0238 Ascidian 4 ACENV0239 Sponge 20 ACENV0240 Sponge 12 ACENV0241 Sponge 23 ACENV0242 Bryozoan 17 ACENV0243 Sponge 9 ACENV0244 Ascidian 52 False positive ACENV0246 Sponge 11 ACENV0247 Sponge 20 ACENV0248 Sponge 7 ACENV0249 Sponge 47 False positive ACENV0250 Ascidian 30 ACENV0251 Sponge 29 ACENV0252 Sponge -6 ACENV0253 Sponge 21 ACENV0254 Sponge 16 ACENV0255 Sponge 13 ACENV0256 Sponge 26 ACENV0257 Sponge 64 False positive ACENV0258 Sponge Toxic Active Active ACENV0259 Sponge 28 ACENV0260 Ascidian Toxic Active Active ACENV0261 Ascidian 16 ACENV0262 Ascidian 26 ACENV0263 Ascidian Toxic Active Active ACENV0264 Sponge 6 ACENV0265 Sponge 3 ACENV0266 Sponge 9 ACENV0267 Sponge 24 ACENV0268 Sponge 18 ACENV0269 Sponge 16 ACENV0270 Ascidian -12 ACENV0271 Ascidian 15 ACENV0272 Sponge 28 ACENV0273 Sponge 40 Active Inactive ACENV0274 Ascidian 4 ACENV0275 Sponge 21 ACENV0277 Mollusc -1 ACENV0278 Sponge 35 ACENV0279 Sponge 10 ACENV0280 Sponge 27 159

ACENV0281 Sponge 18 ACENV0282 Sponge 28 ACENV0283 Sponge 23 ACENV0284 Mollusc 22 ACENV0285 Ascidian 14 ACENV0286 Sponge 11 ACENV0287 Bryozoan Toxic Toxic ACENV0290 Sponge 10 ACENV0291 Sponge 45 Active Inactive ACENV0292 Sponge 17 ACENV0293 Sponge 23 ACENV0294 Sponge 14 ACENV0295 Sponge 19 ACENV0296 Sponge 11 ACENV0297 Ascidian 5 ACENV0298 Sponge 29 ACENV0299 Sponge 21 ACENV0300 Ascidian 15 ACENV0301 Ascidian 14 ACENV0303 Sponge 27 ACENV0305 Sponge -1 ACENV0306 Ascidian 16 ACENV0307 Sponge 16 ACENV0308 Sponge 15 ACENV0309 Ascidian 22 ACENV0310 Sponge 23 ACENV0311 Ascidian 16 ACENV0312 Ascidian 3 ACENV0313 Mollusc 33 ACENV0315 Ascidian 46 Active Inactive ACENV0316 Ascidian -1 ACENV0317 Ascidian 34 ACENV0318 Sponge 9 ACENV0319 Sponge 41 Active Inactive ACENV0320 Sponge 22 ACENV0321 Sponge 16 ACENV0322 Sponge 32 ACENV0323 Ascidian 26 ACENV0324 Sponge 19 ACENV0325 Ascidian 31 ACENV0327 Ascidian 31 ACENV0328 Sponge 13 ACENV0329 Ascidian 22 ACENV0330 Sponge 13 ACENV0331 Sponge -14 ACENV0332 Sponge 24 ACENV0333 Sponge 1 ACENV0334 Sponge 38 160

ACENV0335 Sponge 43 False positive ACENV0336 Sponge 45 False positive ACENV0337 Sponge Toxic Toxic ACENV0339 Sponge 18 ACENV0340 Sponge 18 ACENV0341 Ascidian Toxic Active Active ACENV0343 Sponge 14 ACENV0344 Sponge 15 ACENV0345 Sponge 9 ACENV0346 Ascidian 12 ACENV0347 Sponge 26 ACENV0348 Ascidian 18 ACENV0349 Sponge 19 ACENV0350 Sponge 29 ACENV0351 Ascidian 22 ACENV0352 Ascidian 6 ACENV0353 Sponge 12 ACENV0354 Sponge 3 ACENV0355 Algae 12 ACENV0356 Sponge 7 ACENV0357 Ascidian 11 ACENV0358 Ascidian -3 ACENV0359 Ascidian -2 ACENV0361 Sponge 18 ACENV0362 Sponge 11 ACENV0363 Ascidian 13 ACENV0366 Sponge 1 ACENV0367 Ascidian 26 ACENV0368 Ascidian Toxic Inactive ACENV0369 Ascidian 9 ACENV0370 Sponge 12 ACENV0371 Ascidian 0 ACENV0372 Sponge 61 False positive ACENV0373 Sponge 13 ACENV0374 Sponge 9 ACENV0375 Ascidian 22 ACENV0376 Sponge 20 ACENV0377 Ascidian 0 ACENV0378 Ascidian Toxic Inactive ACENV0380 Sponge 23 ACENV0381 Sponge 11 ACENV0386 Soft Coral 1 ACENV0387 Sponge 10 ACENV0388 Ascidian 25 ACENV0390 Sponge -11 ACENV0391 Bryozoan 2 ACENV0393 Soft Coral 26 ACENV0394 Sponge -1 161

ACENV0395 Soft Coral Toxic Inactive ACENV0396 Sponge -11 ACENV0397 Sponge -1 ACENV0398 Sponge -14 ACENV0399 Sponge 21 ACENV0400 Sponge -2 ACENV0401 Sponge -6 ACENV0402 Sponge Toxic Inactive ACENV0403 Sponge Toxic Inactive ACENV0405 Sponge 10 ACENV0406 Sponge -4 ACENV0407 Sponge 11 ACENV0408 Sponge 18 ACENV0409 Ascidian 22 ACENV0410 Sponge 1 ACENV0411 Sponge 6 ACENV0412 Sponge -1 ACENV0413 Sponge 16 ACENV0415 Ascidian -4 ACENV0416 Ascidian -3 ACENV0417 Sponge 22 ACENV0418 Sponge 51 Active Inactive ACENV0420 Sponge 21 ACENV0421 Sponge 3 ACENV0422 Sponge -23 ACENV0423 Sponge -5 ACENV0425 Ascidian -16 ACENV0426 Ascidian Toxic Inactive ACENV0427 Ascidian 6 ACENV0428 Ascidian 16 ACENV0429 Ascidian -10 ACENV0430 Ascidian 2 ACENV0431 Ascidian Toxic Inactive ACENV0432 Ascidian 0 ACENV0433 Sponge 15 ACENV0434 Sponge 0 ACENV0435 Sponge 4 ACENV0436 Ascidian 15 ACENV0438 Sponge 0 ACENV0439 Sponge Toxic Inactive ACENV0442 Ascidian -9 ACENV0443 Sponge 21 ACENV0445 Sponge 15 ACENV0446 Sponge 3 ACENV0447 Sponge 7 ACENV0450 Sponge -2 ACENV0451 Sponge 5 ACENV0452 Ascidian -6 162

ACENV0453 Sponge -5 ACENV0454 Sponge -6 ACENV0456 Sponge 17 ACENV0458 Ascidian 3 ACENV0459 Zoanthid -15 ACENV0460 Sponge -13 ACENV0461 Sponge -10 ACENV0462 Ascidian Toxic Inactive ACENV0465 Sponge -9 ACENV0466 Sponge -14 ACENV0468 Sponge 8 ACENV0469 Sponge -6 ACENV0470 Sponge -7 ACENV0471 Ascidian 27 ACENV0472 Sponge -3 ACENV0473 Ascidian -4 ACENV0474 Ascidian 22 ACENV0475 Sponge 4 ACENV0476 Sponge -16 ACENV0477 Sponge -7 ACENV0478 Sponge -26 ACENV0479 Sponge -11 ACENV0480 Sponge 14 ACENV0481 Sponge Toxic Inactive ACENV0482 Sponge -8 ACENV0483 Sponge 25 ACENV0484 Ascidian -17 ACENV0486 Sponge 9 ACENV0487 Soft Coral 0 ACENV0488 Soft Coral 1 ACENV0490 Sponge -5 ACENV0491 Sponge 7 ACENV0492 Sponge 13 ACENV0493 Sponge 22 ACENV0494 Sponge -12 ACENV0495 Sponge 3 ACENV0496 Ascidian 28 ACENV0497 Sponge 1 ACENV0498 Ascidian -3 ACENV0499 Ascidian 8 ACENV0500 Ascidian 5 ACENV0501 Sponge -9 ACENV0502 Sponge -6 ACENV0503 Ascidian 12 ACENV0509 Sponge -7 ACENV0511 Ascidian 6 ACENV0512 Ascidian -8 ACENV0514 Sponge Toxic Inactive 163

ACENV0515 Sponge -2 ACENV0519 Sponge 4 ACENV0520 Sponge 19 ACENV0522 Sponge -4 ACENV0524 Sponge -24 ACENV0525 Sponge 16 ACENV0526 Ascidian 23 ACENV0528 Sponge Toxic Toxic ACENV0529 Ascidian -1 ACENV0530 Ascidian 19 ACENV0531 Soft Coral 27 ACENV0532 Ascidian -8 ACENV0533 Ascidian 1 ACENV0535 Sponge 16 ACENV0536 Soft Coral -4 ACENV0537 Soft Coral 12 ACENV0538 Ascidian 14 ACENV0539 Sponge 10 ACENV0540 Ascidian -4 ACENV0541 Sponge 14 ACENV0542 Ascidian 2 ACENV0544 Sponge 17 ACENV0546 Sponge 11 ACENV0549 Sponge 17 ACENV0550 Sponge 4 ACENV0552 Sponge 41 False positive ACENV0553 Ascidian 16 ACENV0554 Ascidian 0 ACENV0555 Sponge 2 ACENV0557 Ascidian -7 ACENV0559 Sponge -17 ACENV0561 Sponge -1 ACENV0562 Ascidian 9 ACENV0563 Ascidian 9 ACENV0564 Sponge 0 ACENV0566 Ascidian -2 ACENV0567 Ascidian 22 ACENV0569 Sponge 19 ACENV0570 Sponge -9 ACENV0572 Bryozoan 12 ACENV0579 Bryozoan 27 ACENV0580 Bryozoan 47 False positive ACENV0581 Bryozoan -3 ACENV0582 Bryozoan 12 ACENV0583 Bryozoan 23 ACENV0584 Bryozoan -1 ACENV0586 Bryozoan 0 ACENV0587 Bryozoan Toxic Inactive 164

ACENV0588 Bryozoan 8 ACENV0589 Bryozoan 2 ACENV0590 Bryozoan 15 ACENV0591 Bryozoan 17 ACENV0592 Bryozoan 8 ACENV0593 Bryozoan 18 ACENV0594 Bryozoan 23 ACENV0595 Bryozoan -10 ACENV0596 Bryozoan -1 ACENV0598 Bryozoan -7 ACENV0599 Bryozoan 13 ACENV0600 Bryozoan 3 ACENV0601 Bryozoan 10 ACENV0602 Bryozoan 22 ACENV0603 Bryozoan 12 ACENV0604 Bryozoan 7 ACENV0605 Bryozoan 10 ACENV0606 Bryozoan 14 ACENV0607 Bryozoan -5 ACENV0608 Bryozoan 25 ACENV0610 Bryozoan Toxic Toxic ACENV0611 Bryozoan -6 ACENV0612 Bryozoan Toxic Toxic ACENV0613 Bryozoan 1 ACENV0614 Bryozoan 20 ACENV0615 Bryozoan 6 ACENV0616 Bryozoan 19 ACENV0617 Bryozoan -7 ACENV0618 Bryozoan Toxic Inactive ACENV0619 Bryozoan Toxic Toxic ACENV0620 Bryozoan Toxic Inactive ACENV0621 Bryozoan 20 ACENV0622 Bryozoan 19 ACENV0623 Bryozoan 13 ACENV0624 Bryozoan 19 ACENV0625 Bryozoan 9 ACENV0626 Bryozoan Toxic Toxic ACENV0627 Bryozoan 21 ACENV0628 Bryozoan 19 ACENV0629 Bryozoan 7

165

SUPPLEMENTARY DATA - CHAPTER 2:

IN PREPARATION FOR PUBLISHING:

Supplementary Data: Anti-prion Butenolides and Diphenylpropanones from the Australian Ascidian Polycarpa procera

Laurence K Jennings,†,‡ Luke P Robertson,†,‡ Kathryn E. Rudolph,‡ Alan L Munn,┴ Anthony R Carroll*,†,‡,§

†Environmental Futures Research Institute, Griffith University (Gold Coast campus), Parklands Drive, Southport, QLD 4222, AUSTRALIA. ‡School of Environment and Science, Griffith University (Gold Coast campus), Parklands Drive, Southport, QLD 4222, AUSTRALIA. §Griffith Institute for Drug Discovery, Griffith University (Brisbane Innovation Park), Don Young Road, Nathan, QLD 4111, AUSTRALIA. ┴School of Medical Science and Molecular Basis of Disease Program, Menzies Health Institute Queensland, Griffith University (Gold Coast campus), Parklands Drive, Southport, QLD 4222, AUSTRALIA. * [email protected]

166

Contents:

1 Figure S1-1: H NMR spectrum (500 MHz) of 1 in DMSO-d6

13 Figure S1-2: C NMR spectrum (125 MHz) of 1 in DMSO-d6

Figure S1-3: COSY NMR spectrum (500 MHz) of 1 in DMSO-d6

Figure S1-4: HSQC NMR spectrum (500 MHz) of 1 in DMSO-d6

Figure S1-5: HMBC NMR spectrum (500 MHz) of 1 in DMSO-d6

Figure S1-6: HMBC NMR expansion (500 MHz) of 1 in DMSO-d6 Figure S1-7: (-) HRESIMS of 1

1 Figure S1-8: H NMR spectrum (500 MHz) of 1 in DMSO-d6 under basic conditions

13 Figure S1-9: C NMR spectrum (125 MHz) of 1 in DMSO-d6 under basic conditions

Figure S1-10: COSY NMR spectrum (500 MHz) of 1 in DMSO-d6 under basic conditions

Figure S1-11: HSQC NMR spectrum (500 MHz) of 1 in DMSO-d6 under basic conditions

Figure S1-12: HMBC NMR spectrum (500 MHz) of 1 in DMSO-d6 under basic conditions

1 Figure S2-1: H NMR spectrum (500 MHz) of 2 in DMSO-d6

13 Figure S2-2: C NMR spectrum (200 MHz) of 2 in DMSO-d6

Figure S2-3: COSY NMR spectrum (500 MHz) of 2 in DMSO-d6

Figure S2-4: HSQC NMR spectrum (500 MHz) of 2 in DMSO-d6

Figure S2-5: HMBC NMR spectrum (500 MHz) of 2 in DMSO-d6 Figure S2-6: (-) HRESIMS of 2

1 Figure S3-1: H NMR spectrum (500 MHz) of 3 in DMSO-d6

13 Figure S3-2: C NMR spectrum (125 MHz) of 3 in DMSO-d6

Figure S3-3: COSY NMR spectrum (500 MHz) of 3 in DMSO-d6

Figure S3-4: HSQC NMR spectrum (500 MHz) of 3 in DMSO-d6

Figure S3-5: HMBC NMR spectrum (500 MHz) of 3 in DMSO-d6 Figure S3-6: (-) HRESIMS of 3

1 Figure S4-1: H NMR spectrum (500 MHz) of 4 in DMSO-d6 167

13 Figure S4-2: C NMR spectrum (125 MHz) of 4 in DMSO-d6

Figure S4-3: COSY NMR spectrum (500 MHz) of 4 in DMSO-d6

Figure S4-4: HSQC NMR spectrum (500 MHz) of 4 in DMSO-d6

Figure S4-5: HMBC NMR spectrum (500 MHz) of 4 in DMSO-d6 Figure S4-6: (-) HRESIMS of 4

1 Figure S5-1: H NMR spectrum (500 MHz) of 5 and 3 in DMSO-d6

Figure S5-2: COSY NMR spectrum (500 MHz) of 5 and 3 in DMSO-d6

Figure S5-3: HSQC NMR spectrum (500 MHz) of 5 and 3 in DMSO-d6

Figure S5-4: HMBC NMR spectrum (500 MHz) of 5 and 3 in DMSO-d6 Figure S5-5: (-) HRESIMS of 5

1 Figure S6-1: H NMR spectrum (500 MHz) of 6 in DMSO-d6

13 Figure S6-2: C NMR spectrum (125 MHz) of 6 in DMSO-d6

Figure S6-3: COSY NMR spectrum (500 MHz) of 6 in DMSO-d6

Figure S6-4: HSQC NMR spectrum (500 MHz) of 6 in DMSO-d6

Figure S6-5: HMBC NMR spectrum (500 MHz) of 6 in DMSO-d6 Figure S6-6: (-) HRESIMS of 6

1 Figure S7-1: H NMR spectrum (800 MHz) of 7 in DMSO-d6

13 Figure S7-2: C NMR spectrum (200 MHz) of 7 in DMSO-d6

Figure S7-3: COSY NMR spectrum (800 MHz) of 7 in DMSO-d6

Figure S7-4: HSQC NMR spectrum (800 MHz) of 7 in DMSO-d6

Figure S7-5: HMBC NMR spectrum (800 MHz) of 7 in DMSO-d6 Figure S7-6: (-) HRESIMS of 7

1 Figure S7-7: H NMR spectrum (800 MHz) of 7 in DMSO-d6 under basic conditions

Figure S7-8: COSY NMR spectrum (800 MHz) of 7 in DMSO-d6 under basic conditions

Figure S7-9: HSQC NMR spectrum (800 MHz) of 7 in DMSO-d6 under basic conditions

Figure S7-10: HMBC NMR spectrum (800 MHz) of 7 in DMSO-d6 under basic conditions 168

O OH O Br Br MeO OH Br

1 Figure 1-1: H NMR spectrum (500 MHz) of 1 in DMSO-d6

13 Figure 1-2: C NMR spectrum (125 MHz) of 1 in DMSO-d6

169

Figure 1-3: COSY NMR spectrum (500 MHz) of 1 in DMSO-d6

Figure 1-4: HSQC NMR spectrum (500 MHz) of 1 in DMSO-d6

170

Figure S1-5: HMBC NMR spectrum (500 MHz) of 1 in DMSO-d6

Figure S1-6: HMBC NMR expansion (500 MHz) of 1 in DMSO-d6

171

Figure S1-7: (-) HRESIMS of 1

1 Figure S1-8: H NMR spectrum (500 MHz) of 1 in DMSO-d6 under basic conditions

172

13 Figure S1-9: C NMR spectrum (125 MHz) of 1 in DMSO-d6 under basic conditions

Figure S1-10: COSY NMR spectrum (500 MHz) of 1 in DMSO-d6 under basic conditions 173

Figure S1-11: HSQC NMR spectrum (500 MHz) of 1 in DMSO-d6 under basic conditions

Figure S1-12: HMBC NMR spectrum (500 MHz) of 1 in DMSO-d6 under basic conditions 174

O OH Br O

Br OH Br O Br

1 Figure S2-1: H NMR spectrum (800 MHz) of 2 in DMSO-d6

13 Figure S2-2: C NMR spectrum (200 MHz) of 2 in DMSO-d6 NOTE: Intense impurity

peaks are from TFA addition 175

Figure S2-3: COSY NMR spectrum (800 MHz) of 2 in DMSO-d6

Figure S2-4: HSQC NMR spectrum (800 MHz) of 2 in DMSO-d6

176

Figure S2-5: HMBC NMR spectrum (800 MHz) of 2 in DMSO-d6

Figure S2-6: (-) HRESIMS of 2

177

O MeO O OMe

Br Br OH

1 Figure S3-1: H NMR spectrum (500 MHz) of 3 in DMSO-d6

13 Figure S3-2: C NMR spectrum (125 MHz) of 3 in DMSO-d6

178

Figure S3-3: COSY NMR spectrum (500 MHz) of 3 in DMSO-d6

Figure S3-4: HSQC NMR spectrum (500 MHz) of 3 in DMSO-d6

179

Figure S3-5: HMBC NMR spectrum (500 MHz) of 3 in DMSO-d6

Figure S3-6: (-) HRESIMS of 3

180

Br O MeO O OMe

Br Br OH

1 Figure S4-1: H NMR spectrum (500 MHz) of 4 in DMSO-d6

13 Figure S4-2: C NMR spectrum (125 MHz) of 4 in DMSO-d6

181

Figure S4-3: COSY NMR spectrum (500 MHz) of 4 in DMSO-d6

Figure S4-4: HSQC NMR spectrum (500 MHz) of 4 in DMSO-d6

182

Figure S4-5: HMBC NMR spectrum (500 MHz) of 4 in DMSO-d6

Figure S4-6: (-) HRESIMS of 4

183

HO MeO O

Br Br Br OH

1 Figure S5-1: H NMR spectrum (500 MHz) of 5 in DMSO-d6

13 Figure S5-2: C NMR spectrum (125 MHz) of 5 in DMSO-d6

184

Figure S5-3: COSY NMR spectrum (500 MHz) of 5 in DMSO-d6

Figure S5-4: HSQC NMR spectrum (500 MHz) of 5 in DMSO-d6

185

Figure S5-5: HMBC NMR spectrum (500 MHz) of 5 in DMSO-d6

Figure S5-6: (-) HRESIMS of 5

186

Br HO MeO O

Br Br Br OH

1 Figure S6-1: H NMR spectrum (800 MHz) of 6 in DMSO-d6

13 Figure S6-2: C NMR spectrum (200 MHz) of 6 in DMSO-d6

187

Figure S6-3: COSY NMR spectrum (800 MHz) of 6 in DMSO-d6

Figure S6-4: HSQC NMR spectrum (800 MHz) of 6 in DMSO-d6

188

Figure S6-5: HMBC NMR spectrum (800 MHz) of 6 in DMSO-d6

Figure S6-6: (-) HRESIMS of 6

189

1 Figure S6-7: H NMR spectrum (800 MHz) of 6 in DMSO-d6 under basic conditions

Figure S6-8: COSY NMR spectrum (800 MHz) of 6 in DMSO-d6 under basic conditions 190

Figure S6-9: HSQC NMR spectrum (800 MHz) of 6 in DMSO-d6 under basic conditions

Figure S6-10: HMBC NMR spectrum (800 MHz) of 6 in DMSO-d6 under basic conditions

191

SUPPLEMENTARY DATA - CHAPTER 3

IN PREPARATION FOR PUBLISHING:

Supplementary Data: New Anti-prion and α-synuclein Aggregation Inhibitory Sterols from the Australian Ascidian, Didemnum sp.

Laurence K. Jennings,†,‡ Dale W. Prebble,†,‡ Mingming Xu,‡,§ Alan L. Munn,┴ George D. Mellick,‡,§ Anthony R. Carroll*,†,‡,§

†Environmental Futures Research Institute, Griffith University (Gold Coast campus),

Parklands Drive, Southport, QLD 4222, AUSTRALIA.

‡School of Environment and Science, Griffith University (Gold Coast campus),

Parklands Drive, Southport, QLD 4222, AUSTRALIA.

§Griffith Institute for Drug Discovery, Griffith University (Brisbane Innovation Park),

Don Young Road, Nathan, QLD 4111, AUSTRALIA.

┴School of Medical Science and Molecular Basis of Disease Program, Menzies Health

Institute Queensland, Griffith University (Gold Coast campus), Parklands Drive,

Southport, QLD 4222, AUSTRALIA.

* [email protected]

192

Contents: 1 Figure S1-1: H NMR spectrum (800 MHz) of 1-3 mixture in DMSO-d6

13 Figure S1-2: C NMR spectrum (200 MHz) of 1-3 mixture in DMSO-d6

Figure S1-3: COSY NMR spectrum (800 MHz) of 1-3 mixture in DMSO-d6

Figure S1-4: COSY NMR expansion of 1-3 mixture in DMSO-d6

Figure S1-5: TOCSY NMR spectrum (800 MHz) of 1-3 mixture in DMSO-d6

Figure S1-6: TOCSY NMR expansion of 1-3 mixture in DMSO-d6

Figure S1-7: HSQC NMR spectrum (800 MHz) of 1-3 mixture in DMSO-d6

Figure S1-8: HMBC NMR spectrum (800 MHz) of 1-3 mixture in DMSO-d6

Figure S1-9: HMBC NMR expansion of 1-3 mixture in DMSO-d6

Figure S1-10: ROESY NMR spectrum (800 MHz) of 1-3 mixture in DMSO-d6

1 Figure S2-1: H NMR spectrum (800 MHz) of 1 in DMSO-d6

1 Figure S2-2: H NMR expansion of 1 in DMSO-d6

13 Figure S2-3: C NMR spectrum (200 MHz) of 1 in DMSO-d6

Figure S2-4: COSY NMR spectrum (800 MHz) of 1 in DMSO-d6

Figure S2-5: COSY NMR expansion of 1 in DMSO-d6

Figure S2-6: HSQC NMR spectrum (800 MHz) of 1 in DMSO-d6

Figure S2-7: HMBC NMR spectrum (800 MHz) of 1 in DMSO-d6

Figure S2-8: HMBC NMR expansion of 1 in DMSO-d6

Figure S2-9: HMBC NMR expansion of 1 in DMSO-d6

Figure S2-10: ROESY NMR spectrum (800 MHz) of 1 in DMSO-d6

1 Figure S3-1: H NMR spectrum (800 MHz) of 2 in DMSO-d6

1 Figure S3-2: H NMR expansion of 2 in DMSO-d6

Figure S3-3: COSY NMR spectrum (800 MHz) of 2 in DMSO-d6

Figure S3-4: COSY NMR expansion of 2 in DMSO-d6

Figure S3-5: HSQC NMR spectrum (800 MHz) of 2 in DMSO-d6

193

Figure S3-6: HMBC NMR spectrum (800 MHz) of 2 in DMSO-d6

1 Figure S4-1: H NMR spectrum (800 MHz) of 3 in DMSO-d6

1 Figure S4-2: H NMR expansion of 2 in DMSO-d6

Figure S4-3: COSY NMR spectrum (800 MHz) of 3 in DMSO-d6

Figure S4-4: COSY NMR expansion of 3 in DMSO-d6

Figure S4-5: HSQC NMR spectrum (800 MHz) of 3 in DMSO-d6

Figure S4-6: HMBC NMR spectrum (800 MHz) of 3 in DMSO-d6

194

18 R 11 H 19 16 1 9 H 14

3 5 H 7 OH HO OH H

1 Figure S1-1: H NMR spectrum (800 MHz) of 1-3 mixture in DMSO-d6

13 Figure S1-2: C NMR spectrum (200 MHz) of 1-3 mixture in DMSO-d6

195

Figure S1-3: COSY NMR spectrum (800 MHz) of 1-3 mixture in DMSO-d6

Figure S1-4: COSY NMR expansion of 1-3 mixture in DMSO-d6

196

Figure S1-5: TOCSY NMR spectrum (800 MHz) of 1-3 mixture in DMSO-d6

Figure S1-6: TOCSY NMR expansion of 1-3 mixture in DMSO-d6

197

Figure S1-7: HSQC NMR spectrum (800 MHz) of 1-3 mixture in DMSO-d6

Figure S1-8: HMBC NMR spectrum (800 MHz) of 1-3 mixture in DMSO-d6

198

Figure S1-9: HMBC NMR expansion of 1-3 mixture in DMSO-d6

Figure S1-10: ROESY NMR spectrum (800 MHz) of 1-3 mixture in DMSO-d6

199

21 22 18 24 26 11 H 19 16 OSO H 1 9 H 3 14

3 5 H 7 OH HO OH H

1 Figure S2-1: H NMR spectrum (800 MHz) of 1 in DMSO-d6

1 Figure S2-2: H NMR expansion of 1 in DMSO-d6

200

13 Figure S2-3: C NMR spectrum (200 MHz) of 1 in DMSO-d6

Figure S2-4: COSY NMR spectrum (800 MHz) of 1 in DMSO-d6

201

Figure S2-5: COSY NMR expansion of 1 in DMSO-d6

Figure S2-6: HSQC NMR spectrum (800 MHz) of 1 in DMSO-d6

202

Figure S2-7: HMBC NMR spectrum (800 MHz) of 1 in DMSO-d6

Figure S2-8: HMBC NMR expansion (800 MHz) of 1 in DMSO-d6

203

Figure S2-9: HMBC NMR expansion of 1 in DMSO-d6

Figure S2-10: ROESY NMR spectrum (800 MHz) of 1 in DMSO-d6

204

21 22 18 24 26 11 H 19 16 OSO H 1 9 H 3 14

3 5 H 7 OH HO OH H

1 Figure S3-1: H NMR spectrum (800 MHz) of 2 in DMSO-d6

1 Figure S3-2: H NMR expansion of 2 in DMSO-d6

205

Figure S3-3: COSY NMR spectrum (800 MHz) of 2 in DMSO-d6

Figure S3-4: COSY NMR expansion of 2 in DMSO-d6

206

Figure S3-5: HSQC NMR spectrum (800 MHz) of 2 in DMSO-d6

Figure S3-6: HMBC NMR spectrum (800 MHz) of 2 in DMSO-d6

207

21 28 22 18 26 11 H 19 16 OSO H 1 9 H 3 14 27

3 5 H 7 OH HO OH H

1 Figure S4-1: H NMR spectrum (800 MHz) of 3 in DMSO-d6

1 Figure S4-2: H NMR expansion of 2 in DMSO-d6

208

Figure S4-3: COSY NMR spectrum (800 MHz) of 3 in DMSO-d6

Figure S4-4: COSY NMR expansion of 3 in DMSO-d6

209

Figure S4-5: HSQC NMR spectrum (800 MHz) of 3 in DMSO-d6

Figure S4-6: HMBC NMR spectrum (800 MHz) of 3 in DMSO-d6.

210

SUPPLEMENTARY DATA - CHAPTER 4

Contents:

Figure S1-1: 1H NMR spectrum (800 MHz) of a mixture of Dysidea-sterol A-11- acetate (1) and Dysidea-sterol B-11-acetate (2) in DMSO-d6. Figure S1-2: 13C NMR spectrum (200 MHz) of a mixture of Dysidea-sterol A-11- acetate (1) and Dysidea-sterol B-11-acetate (2) in DMSO-d6. Figure S1-5: COSY NMR spectrum (800 MHz) of a mixture of Dysidea-sterol A-11- acetate (1) and Dysidea-sterol B-11-acetate (2) in DMSO-d6. Figure S1-6: HSQC NMR spectrum (800 MHz) of a mixture of Dysidea-sterol A-11- acetate (1) and Dysidea-sterol B-11-acetate (2) in DMSO-d6. Figure S1-7: HMBC NMR spectrum (800 MHz) of a mixture of Dysidea-sterol A-11- acetate (1) and Dysidea-sterol B-11-acetate (2) in DMSO-d6. Figure S1-8: ROESY NMR spectrum (800 MHz) of a mixture of Dysidea-sterol A-11- acetate (1) and Dysidea-sterol B-11-acetate (2) in DMSO-d6.

Figure S2-1: 1H NMR spectrum (800 MHz) of a mixture of Dysidea-sterol A-11,19- diacetate (3) and Dysidea-sterol B-11,19-diacetate (4) in DMSO-d6. Figure S2-2: 13C NMR spectrum (200 MHz) of a mixture of Dysidea-sterol A-11,19- diacetate (3) and Dysidea-sterol B-11,19-diacetate (4) in DMSO-d6. Figure S2-5: COSY NMR spectrum (800 MHz) of a mixture of Dysidea-sterol A-

11,19-diacetate (3) and Dysidea-sterol B-11,19-diacetate (4) in DMSO-d6. Figure S2-6: HSQC NMR spectrum (800 MHz) of a mixture of Dysidea-sterol A-

11,19-diacetate (3) and Dysidea-sterol B-11,19-diacetate (4) in DMSO-d6. Figure S2-7: HMBC NMR spectrum (800 MHz) of a mixture of Dysidea-sterol A-

11,19-diacetate (3) and Dysidea-sterol B-11,19-diacetate (4) in DMSO-d6. Figure S2-8: ROESY NMR spectrum (800 MHz) of a mixture of Dysidea-sterol A-

11,19-diacetate (3) and Dysidea-sterol B-11,19-diacetate (4) in DMSO-d6.

211

AcO H HO OH HO H HO OH OH 1

AcO H HO OH HO H HO OH OH 2

Figure S1-1: 1H NMR spectrum (800 MHz) of a mixture of Dysidea-sterol A-11-acetate (1) and Dysidea-sterol B-11-acetate (2) in DMSO-d6.

Figure S1-2: 13C NMR spectrum (200 MHz) of a mixture of Dysidea-sterol A-11- acetate (1) and Dysidea-sterol B-11-acetate (2) in DMSO-d6.

212

Figure S1-3: COSY spectrum (800 MHz) of a mixture of Dysidea-sterol A-11-acetate (1) and Dysidea-sterol B-11-acetate (2) in DMSO-d6.

Figure S1-4: HSQC spectrum (800 MHz) of a mixture of Dysidea-sterol A-11-acetate (1) and Dysidea-sterol B-11-acetate (2) in DMSO-d6.

213

Figure S1-5: HMBC spectrum (800 MHz) of a mixture of Dysidea-sterol A-11-acetate (1) and Dysidea-sterol B-11-acetate (2) in DMSO-d6.

Figure S1-6: ROESY spectrum (800 MHz) of a mixture of Dysidea-sterol A-11-acetate (1) and Dysidea-sterol B-11-acetate (2) in DMSO-d6.

214

AcO H AcO OH HO H HO OH OH 3

AcO H AcO OH HO H HO OH OH 4

Figure S2-1: 1H NMR spectrum (800 MHz) of a mixture of Dysidea-sterol A-11,19- diacetate (3) and Dysidea-sterol B-11,19-diacetate (4) in DMSO-d6.

Figure S2-2: 13C NMR spectrum (200 MHz) of a mixture of Dysidea-sterol A-11,19- diacetate (3) and Dysidea-sterol B-11,19-diacetate (4) in DMSO-d6.

215

Figure S2-3: COSY spectrum (800 MHz) of a mixture of Dysidea-sterol A-11,19- diacetate (3) and Dysidea-sterol B-11,19-diacetate (4) in DMSO-d6.

Figure S2-4: HSQC spectrum (800 MHz) of a mixture of Dysidea-sterol A-11,19- diacetate (3) and Dysidea-sterol B-11,19-diacetate (4) in DMSO-d6.

216

Figure S2-5: HMBC spectrum (800 MHz) of a mixture of Dysidea-sterol A-11,19- diacetate (3) and Dysidea-sterol B-11,19-diacetate (4) in DMSO-d6.

Figure S2-6: ROESY spectrum (800 MHz) of a mixture of Dysidea-sterol A-11,19- diacetate (3) and Dysidea-sterol B-11,19-diacetate (4) in DMSO-d6.

217

SUPPLEMENTARY DATA - CHAPTER 5

Table S5-1. ClogP and tPSA calculations of the natural products isolated in this study. Species Natural products MW cLogP tPSA S. ianthelliformis Aplysamine-1 (1) 408.2 3.9 15.7 Aplysamine-2 (2) 650.2 6.1 83.4 Purealidine-Q (3) 745.1 5.9 92.6 P. procera Procerolide A (4) 549.0 5.2 76.0 Procerolide B (5) 626.0 5.9 76.0 Procerolide C (6) 563.0 5.3 65.0 Procerolide D (7) 641.9 6.0 65.0 Procerone A (9) 509.0 4.4 66.8 Procerone B (10) 587.9 5.1 66.8 Didemnum sp. Didemnisterol A (11) 516.7 4.6 124.3 Didemnisterol B (12) 514.7 4.2 124.3 Didemnisterol C (13) 528.7 4.6 124.3 Dysidea sp. Dysidea-sterol 11-acetate (14) 524.7 3.4 147.7 Dysidea-sterol 11,19-diacetate (15) 566.7 4.2 153.8 Table S5-2. ClogP and tPSA calculations of the anti-prion leads in the Introduction. Class Anti-prion leads MW cLogP tPSA Sulfated polyanionic 1 602.0 -5.4 333.9 glycans (monoisotopic) 2 417.3 -4.1 249.0 3 582.5 -7.0 305.9 4 594.4 -6.7 357.2 Sulfated azo dye 5 650.7 1.3 215.0 derivatives 6 478.7 -3.2 32.3 7 900.8 -7.2 389.2 8 368.4 2.3 93.1 9 1297.3 -8.6 483.8 10 248.3 0.9 86.2 Cyclic tetrapyrroles 11 616.7 2.6 204.3 12 678.8 -11.3 60.8 13 830.8 -5.3 266.3 14 78.1 -1.4 17.1 15 92.1 -1.5 60.7 16 75.1 0.2 23.1 Polyphenolic derivatives 17 1707.2 < -12.0 778.0 18 866.8 0.0 331.1 19 458.4 1.5 197.4 20 610.5 2.3 264.1 21 304.3 0.3 130.6 22 254.2 3.6 66.8 Phenothiazine and 23 400.0 6.7 36.9 quinoline derivatives 24 319.9 5.1 27.6 25 320.9 5.1 6.5 26 324.4 2.8 45.1 218

27 194.2 2.9 38.4 28 198.3 3.3 38.9 29 426.3 6.4 60.8 30 631.6 9.7 85.7 Poly-aromatic PrPC 31 306.3 3.5 81.9 binding compounds 32 343.2 5.3 42.9 33 231.1 3.0 74.3 34 330.3 2.9 73.8 35 333.4 2.5 121.2 36 445.3 1.4 109.3 37 420.6 3.8 64.7 38 299.3 3.4 77.2 Polyamine compounds 39 1443.9 < -12.0 576.8 (Dendrimers are Gen. 1) 40 781.4 < -12.0 240.6 41 2784.8 < -12.0 328.3 42 493.6 4.4 84.7 Tetracyclines 43 653.4 1.9 185.8 44 444.4 -0.9 181.6 45 444.4 -2.8 181.6 Cholesterol Inhibitors 46 924.1 -3.7 319.6 47 654.8 2.9 208.4 48 1174.3 < -12.0 381.6 49 404.5 4.1 72.8 50 418.6 4.5 72.8 51 688.8 2.6 214.2 52 454.6 4.5 64.0 Chelators of redox-active 53 149.2 -1.7 63.3 metal 54 305.5 3.7 32.6 55 252.3 0.2 84.6 56 208.3 3.0 24.7 57 360.5 6.8 24.7 58 256.3 4.3 24.7 Table S5-3. ClogP and tPSA calculations of the currently used CNS drugs. CNS drugs MW cLogP tPSA Acamprosate 181.2 -2.5 83.5 Alprazolam 308.8 2.6 43.1 Amfebutamone 239.7 3.2 29.1 Amisulpride 369.5 1.8 101.7 Amphetamine 135.2 1.7 26 Aniracetam 219.2 0.7 46.6 Apomorphine 267.3 2.5 43.7 Aprepitant 534.4 4.6 83.2 Aripiprazole 448.4 5.3 44.8 Atomoxetine 255.4 3.9 21.3 Bromazepam 316.2 1.7 54.4 Bromocriptine 654.6 6.6 118.2 Brotizolam 393.7 2.7 43.1

219

Budipine 293.4 5.6 3.2 Buprenorphine 467.6 4 62.2 Buspirone 385.5 2.2 69.6 Cabergoline 451.6 4.2 71.7 Caffeine 194.2 0 61.8 Carbamazepine 236.3 2.4 46.3 Carisoprodol 260.3 2.3 90.7 Chlorpromazine 318.9 5.3 6.5 Citalopram 324.4 3.1 36.3 Clomipramine 314.9 5.9 6.5 Clonazepam 315.7 2.4 84.6 Clozapine 326.8 3.7 30.9 Cyclobenzaprine 275.4 5.1 3.2 Dexmethylphenidate 233.3 2.6 38.3 Dextropropoxyphene 339.5 5.2 29.5 Diazepam 284.7 3 32.7 Donepezil 379.5 4.6 38.8 Dronabinol 314.5 7.2 29.5 Duloxetine 297.4 4.3 21.3 Eletriptan 382.5 3.4 53.2 Escitalopram 324.4 3.1 36.3 Eszopiclone 388.8 1.3 91.8 Ethosuximide 141.2 0.4 46.2 Felbamate 238.2 0.5 104.6 Fentanyl 336.5 3.6 23.6 Flumazenil 303.3 1.3 64.4 Fluoxetine 309.3 4.6 21.3 Fluvoxamine 318.3 3.3 56.8 Gabapentin 171.2 -0.7 63.3 Galantamine 287.4 1 41.9 Haloperidol 375.9 3.8 40.5 Hydrocodone 299.4 1.1 38.8 Indeloxazine 231.3 2.2 30.5 Lamotrigine 256.1 2.5 90.7 Levetiracetam 170.2 -0.3 63.4 Levomethadyl Acetate 353.5 4.6 29.5 Lofexidine 259.1 3.5 33.6 Lorazepam 321.2 2.4 61.7 Meprobamate 218.3 0.9 104.6 Methylphenidate 233.3 2.6 38.3 Metoclopramide 299.8 2.2 67.6 Midazolam 325.8 3.4 30.2 Milnacipran 246.3 1.9 46.3 Minaprine 298.4 3.2 50.3 Mirtazapine 265.4 2.8 19.4 Moclobemide 268.7 2.2 41.6 Modafinil 273.4 0.9 60.2 Morphine 285.3 0.6 52.9 220

Nalmefene 339.4 2.6 52.9 Naltrexone 341.4 0.4 70 Nemonapride 387.9 4.2 53.6 Nicergoline 484.4 4.1 56.6 Nicotrol 162.2 0.9 16.1 Nimodipine 418.4 4 117 Nortriptyline 263.4 4.3 12 Olanzapine 312.4 3 30.9 Oxcarbazepine 252.3 1.2 63.4 Oxycodone 315.4 0 59 Paliperidone 426.5 1.1 84.4 Paroxetine 329.4 4.2 39.7 Pergolide 314.5 4.4 19 Perospirone 426.6 3.8 56.8 phenytoin 252.3 2.1 58.2 Piracetam 142.2 -1.2 63.4 Pramipexole 211.3 1.2 50.9 Pregabalin 159.2 -0.9 63.3 Propofol 178.3 3.9 20.2 Propoxyphene 339.5 5.2 29.5 Quazepam 386.8 3.2 15.6 Quetiapine 383.5 3 48.3 Ramelteon 259.3 2.5 38.3 Rasagiline 171.2 2.5 12 Reboxetine 313.4 3.3 39.7 Remifentanil 376.4 2 76.2 Riluzole 234.2 3.2 48.1 Rimonabant 463.8 6.5 50.2 Risperidone 410.5 2.7 64.2 Rivastigmine 250.3 2.1 32.8 Ropinirole 260.4 2.8 32.3 Ropivacaine 274.4 3.2 32.3 Selegiline 187.3 3 3.2 Sertindole 440.9 5.3 40.5 Sertraline 306.2 5.3 12 Sinequan 279.4 4.1 12.5 Sulpiride 341.4 1.1 101.7 Tacrine 198.3 3.3 38.9 Talipexole 209.3 1.1 42.2 Terguride 340.5 2.8 51.4 Thiopental 242.3 1.8 58.5 Tiagabine 375.5 2.8 40.5 Tianeptine 437 1.5 86.7 Topiramate 339.4 0 115.5 Tramadol 263.4 3.1 32.7 Trazodone 371.9 3.9 45.8 Tropisetron 284.4 2.9 45.3 Valproic Acid 144.2 2.8 37.3 221

Varenicline 211.3 0.9 37.8 Venlafaxine 277.4 3.3 32.7 Verapamil 454.6 4.5 64 Vigabatrin 129.2 -2.2 63.3 Vinpocetine 350.5 4.8 34.5 Zaleplon 305.3 1.4 74.3 Ziprasidone 412.9 4.2 48.5 Zolpidem 307.4 3 37.6 Zonisamide 212.2 -0.4 86.2 Zotepine 331.9 5.1 12.5 Zotepine 331.9 5.1 12.5 NOTE: The list of CNS drugs was acquired from Wager, T. T.; Hou, X.; Verhoest, P. R.; Villalobos, A. ACS chemical neuroscience 2010, 1, 435-449.

222