Exploring marine sponges as a source of novel chemical entities for drug development A thesis submitted for the degree of Philosophiae Doctor (PhD) by Christopher Martin Hatton, MPharm Cardiff School of Pharmacy and Pharmaceutical Science Cardiff University July 2015 Declaration Declaration i Acknowledgments Acknowledgments I must give the greatest thanks to my supervisors Dr Alex White, Professor Les Baillie and Dr David Rooke for their insight, guidance and friendship during my three years as a postgraduate student. I would like to pay particular thanks to Alex for his support and encouragement on a daily basis. I must also thank Dr Anneke Lubben (Bath University) for her assistance in the completion of HPLC-MS/MS and Dr Julian Marchesi for his facilitation of the DNA analysis of samples. Particular thanks goes to Dr Svetlana Ignatova and Dr Peter Hewitson (Brunel University) for their advice and assistance with the use of HPCCC. Further thanks goes to the Archipelagos research group including Dr Rupert Perkins who facilitated the collection trip to Greece and the SeaSearch group in particular Dr Claire Goodwin, Dr Jennifer Jones and Kate Lock for aid with local species lists, collection and identification of marine sponges. I must also thank the Knowledge Economy Skills Scholarships (KESS) group for funding this project alongside my industrial sponsor Sarum Biosciences. I would additionally like to thank the members of the natural products and microbiology groups and others within Cardiff University for their support, advice and most importantly their attempts of humour throughout the course of my studies. Finally I would like to thank my mother and father, partner Christina and the rest of my family and friends for their continued support and encouragement in every challenge I undertake. ii Acknowledgments “They may look like plants but sponges are one of the simplest of all living animals, yet in their own way they are amazing” -Sir David Attenborough Attenborough’s Ark, Natural World SpeCial, BBC, 5/11/2012 iii Abstract Abstract Antibacterial resistant infections are one of the most challenging problems affecting healthcare and have developed through the overuse of antibiotics and a shortage of new treatments progressing to market. Natural products are the initial source of most antibiotics currently available and marine sponges are a known resource of novel antibacterial compounds; although well-studied marine sponges found in UK waters have been scarcely explored. An examination of the chemical research on sponges identified previously unstudied species for collection in both Greece and Wales. Sequential solvent gradient extraction was optimised, to best exploit the material collected, providing three crude extracts for each sponge collected. A significant difference was observed between the chemical composition of sponges collected from Greece and Wales. An efficient antimicrobial assay was developed to screen each extract against clinically relevant organisms; allowing the direct identification of activity on an eluted thin layer chromatography plate. This overlay data was used for detailed chemical analysis using high performance counter current chromatography, with some separated fractions displaying greater activity towards the bacterium methicillin resistant StaphyloCoCCus aureus (MRSA) than vancomycin. The parent masses of compounds responsible for activity were identified by directly coupling the overlay assay data to mass spectrometry, identifying multiple novel parent masses. Dereplication of samples was completed using the database MarinLit and the construction of a molecular network to compare fragmentation patterns in mass spectra. Bacterial cultivation from Welsh sponge samples isolated 18 antibacterial strains, which were identified using 16S rRNA analysis. Four of these strains were previously uncultured. Chemical analysis was also completed, on two unstudied strains, identifying further active novel parent masses, with no parent mass crossover to the host sponge. Overall, this investigation concluded that marine sponges are excellent source of novel antibacterial compounds, which can display activity against clinically relevant bacteria equivalent to current treatments. iv Table of contents Table of Contents Declaration ..................................................................................................................................... i Abstract ......................................................................................................................................... iv Abbreviations ............................................................................................................................. XIII 1 Introduction .................................................................................................................. 2 1.1 Marine life ........................................................................................................................... 2 1.1.1 Classifications of animals and marine life ...................................................................... 2 1.2 Marine sponges (phylum Porifera) ............................................................................ 4 1.2.1 Types of sponges ....................................................................................................................... 5 1.2.2 Classes of sponge ....................................................................................................................... 7 1.3 The medicinal use of natural products ..................................................................... 8 1.3.1 Current and historical place of natural products in drug therapy ....................... 8 1.3.2 Natural products as source of novel metabolites for drug development ......... 8 1.3.3 Marine life as a source of natural products .................................................................. 10 1.4 Marine sponges as a source of novel metabolites for drug development .. 11 1.4.1 Peptides ....................................................................................................................................... 13 1.4.2 Polyketides ................................................................................................................................. 15 1.4.3 Alkaloids ...................................................................................................................................... 17 1.4.4 Terpenes ..................................................................................................................................... 21 1.4.5 Licensed medicines derived from marine sponges. ................................................. 25 1.4.6 Secondary metabolite biosynthesis: is it the bacteria or the sponge? ............. 26 1.5 ‘Super bugs’ or drug resistant bacteria .................................................................. 29 1.5.1 Classification of bacteria ...................................................................................................... 30 1.5.2 Anti-infective agents .............................................................................................................. 31 1.5.3 Current classes of antibiotics ............................................................................................. 31 1.5.3.1 Protein synthesis inhibitors ....................................................................................................... 31 1.5.3.2 Cell wall synthesis inhibitors ..................................................................................................... 33 1.5.3.3 Nucleic acid synthesis inhibitors .............................................................................................. 33 1.5.4 The development of antibiotic resistance .................................................................... 34 1.5.5 Identifying the need for new antibiotics ....................................................................... 35 1.5.6 The discovery void .................................................................................................................. 39 1.5.7 Addressing the problem of drug-resistant infections ............................................. 41 1.6 Aim and objectives ........................................................................................................ 43 2 Materials and methods ........................................................................................... 45 2.1 Materials ........................................................................................................................... 45 V Table of contents 2.1.1 Reagents and solvents ........................................................................................................... 45 2.1.2 Equipment and glassware ................................................................................................... 45 2.2 Sponge samples .............................................................................................................. 45 2.2.1 Sponge delivery and storage .............................................................................................. 45 2.2.1.1 Greek samples .................................................................................................................................. 45 2.2.1.2 Welsh samples .................................................................................................................................. 46 2.2.2 Processing sponge for