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Cyclotides from an Extreme Habitat: Characterization of Cyclic Peptides from Viola Abyssinica of Ethiopian Highlands

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Cyclotides from an Extreme Habitat: Characterization of Cyclic Peptides from Viola Abyssinica of Ethiopian Highlands To my late grandmother List of Papers This thesis is based on the following papers, which are referred to in the text by their Roman numerals. I Yeshak, MY., Burman, R., Eriksson, C., Göransson, U. Opti- mization of Cyclotide Extraction Parameters. Submitted to Phy- tochemistry Letters. II Yeshak, MY., Burman, R., Asres, K., Göransson, U. (2011) Cyclotides from an Extreme Habitat: Characterization of Cyclic Peptides from Viola abyssinica of Ethiopian Highlands. Journal of Natural Products, 74:727-731. Reprinted with permission. Copyright © 2011 The American Chemical Society and Ameri- can Society of Pharmacognosy. III Yeshak, MY., Strömstedt, A., Burman, R., Göransson, U., Cy- clotide stability in Pseudomonas aeruginosa and Staphylococ- cus aureus Secreted Proteases. In manuscript. IV Yeshak, MY., Göransson, U., Burman, R., Hellman, B., Geno- toxicity and Cellular Uptake of Cyclotides: Evidence for Multi- ple Mode of Action. Accepted. Journal of Mutation Research - Genetic Toxicology and Environmental Mutagenesis. Contents Introduction ...................................................................................................11 Natural products .......................................................................................11 Cyclotides .................................................................................................12 The cyclotide framework..........................................................................13 Natural sources and biosynthesis..............................................................15 Biological activities ..................................................................................16 Potential applications of cyclotides ..........................................................17 Aims of this study.....................................................................................18 Biochemical studies on cyclotides ................................................................19 Extraction and isolation............................................................................19 Small-scale extraction..........................................................................19 Large-scale extraction..........................................................................22 Identification and sequence determination...............................................27 Mass spectrometry ...............................................................................27 Ambiguities to solve ............................................................................29 Amino acid analysis.............................................................................32 Quantitative analysis.................................................................................33 Stability in proteases.................................................................................34 Cyclotides and cells.......................................................................................38 Cytotoxicity ..............................................................................................38 Genotoxicity .............................................................................................40 Cell permeation.........................................................................................44 Radiolabeling .......................................................................................45 Microautoradiographic analysis...........................................................45 Discussion .....................................................................................................50 Cyclotide production ................................................................................50 Nature as a source of novel templates ......................................................51 Diverse sequences................................................................................51 Lessons from the knot..........................................................................53 Multiple modes of action..........................................................................54 Concluding remarks and future perspectives ................................................56 Popular science summary..............................................................................58 አጭር መግለጫ..............................................................................................60 Acknowledgements .......................................................................................66 References .....................................................................................................70 Appendices....................................................................................................80 Appendix I ................................................................................................80 Appendix II...............................................................................................81 Abbreviations 3D three dimensional AAA (quantitative) amino acid analysis AMPs antimicrobial peptides APG Angiosperm phylogeny group AQC 6-amino-quinolyl-N-hydroxysuccinimidyl carbamate ASL above sea level BPI base peak ion C-18 carbon-18 CCK cyclic cystine knot cyO18 cycloviolacin O18 cyO19 cycloviolacin O19 cyO2 cycloviolacin O2 DMSO dimethyl sulfoxide ESI electro spray ionization HPLC high performance liquid chromatography HTS high throughput screening IC50 the concentration that inhibited survival by 50% kB1 kalata B1 LC liquid chromatography MARG microautoradiography MS mass spectrometry MS-MS tandem mass spectrometry PBS phosphate-buffered saline solution PE pseudomonas elastase Q-TOF quadropole time of flight RP reversed phase RPMI Roswell Park Memorial Institute medium SCG single cell gel SCX strong cation exchange chromatography SPE solid phase extraction TC top concentration (corresponding to IC50 in FMCA) TDNA tail DNA Xaa an amino acid Amino acids Amino acid Three letter code One letter code Polarity/Net charge Alanine Ala A hydrophobic Arginine Arg R positively charged Asparagine Asn N hydrophilic Aspartic acid Asp D negatively charged Cysteine Cys C hydrophilic Glutamic acid Glu E negatively charged Glutamine Gln Q hydrophilic Glycine Gly G hydrophobic Histidine His H positively charged Isoleucine Ile I hydrophobic Leucine leu L hydrophobic Lysine Lys K positively charged Methionine Met M hydrophobic Phenylalanine Phe F hydrophobic Proline Pro P hydrophobic Serine Ser S hydrophilic Threonine Thr T hydrophilic Tryptophan Try W hydrophobic Tyrosine Tyr Y hydrophobic Valine Val V hydrophobic Introduction Natural products atural products are chemical compounds derived from plants, animals, N and microorganisms; the term is usually reserved for secondary me- tabolites that are not involved in primary metabolic pathways (Canell 1998; Kinghorn et al., 2011; Pawlik, 2011). The discovery and use of natural prod- ucts by human beings dates back to pre-historic periods (Koehn and Carter, 2005; Pawlik, 2011); natural products have also been an essential source of drug discovery, inspiring scientists for millennia (Clardy and Walsh, 2004; Hong, 2011). They can be utilized for the pharmacological actions they in- duce by their own right or as pharmacophores upon which semi-synthetic or synthetic analogues can be designed (Koehn and Carter, 2005). The lessons synthetic and medicinal chemists take from the architectural platforms of biosynthesis used by nature are also invaluable (Clardy and Walsh, 2004). Natural products are the richest source of novel compound classes for bio- logical/pharmacological studies due to their wide structural and functional diversity, biochemical specificity, and desirable molecular properties (Koehn and Carter, 2005; Shen et al., 2003). About 50% of the currently marketed drugs discovered between the period of 1981 to 2010 owe their origin to natural products or natural product derivatives; the role of natural products is more pronounced in the areas of anti-cancer and anti-infective agents, where almost two-thirds of the drugs are derived from natural products (Newman and Cragg, 2012). Despite this success, the interest of the pharmaceutical industry in natural products for drug discovery has declined during recent years in favor of newer strategies such as high throughput screening (HTS), combinatorial chemistry, and genome mining, and also for commercial rea- sons (Hong, 2011; Kingston, 2011). These strategies first offered a simpler technique of addressing a defined molecular target and optimizing drug-like structures (Carter, 2011; Koehn and Carter, 2005; Newman and Cragg, 2012); however, the expectations of the pharmaceutical industry did not come to realization but rather to a much higher rate of failure than predicted (Carter, 2011; Koehn and Carter, 2005). For instance, in 30 years of time, combinatorial chemistry could provide only one compound approved as a drug, i.e., the antitumor compound known as sorafenib (Newman and Cragg, 2012). On the other hand, the technological advances in isolation and charac- terization, function-oriented synthesis, and biotechnology are addressing such limitations as chemical complexity, difficulty in access, and supply of 11 natural products (Carter, 2011; Clardy and Walsh, 2004; Koehn and Carter, 2005; O'Keefe, 2001). Hence, the field of natural product research is once again prevailing in drug discovery (Carter, 2011; Kingston, 2011). In fact, there are suggestions that for the pharmaceutical industry to improve, what is required is digging into natural product
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