Antimicrobial peptides from the Arctic ascidian Synoicum turgens

The rise in frequency of antibiotic resistant pathogenic bacteria makes the need for new treatment options for previously curable bacterial infections ever more important. In the process of discovering and developing antibacterial agents, one powerful approach has been borrowing wisdom from nature. Antimicrobial peptides (AMPs) are critical components of the innate immune systems found in almost all eukaryotic life forms. Their mode of action includes disruption of the bacterial membrane and to trigger supporting immune responses. Due to these properties, AMPs are considered promising lead structures that can be further developed into commercially available antibiotics to treat or prevent human diseases caused by bacteria. The work conducted in this thesis aims to discover and characterize novel antimicrobial peptides from the Arctic marine ascidian Synoicum turgens by using a marine bioprospecting approach. This includes collection, extraction and fractionation of biomass, antibacterial bioactivity testing and AMP isolation followed by chemical and biological characterization. For one isolated peptide class, truncated versions were prepared, aiming to produce shorter, linear variants with retained antimicrobial activity. In paper I, turgencin A and B and their oxidized derivatives were discovered through bioassay-guided purification. These peptides had an unusual disulfide connectivity, rarely seen in marine AMPs. Both turgencin A and B were potently active against all assayed bacterial strains. Membrane assays showed that the peptides cause bacterial membrane disruption within a few seconds. Turgencin A and B also displayed some cytotoxic activity against two human cell lines. Oxidation of the methionine present in both peptides decreased the bioactivities in all assays. Turgencin A, being the most potent AMP, was subject for sequence analysis and prediction of antimicrobial potential of different regions in paper II. Truncated and modified variants of turgencin A were synthetically produced to make smaller AMPs with the potential of being developed into antimicrobial drug leads. These 10-mer peptides, named StAMP-1–11, were made using an amino acid replacement strategy. Some of the Trp enriched peptides had similar bactericidal activity as the parent peptide turgencin A, and no cytotoxic activity against the mammalian cell lines. During turgencin isolation, a series of other smaller peptides were discovered in the same extracts presumably with antimicrobial activity. These isolated and characterized ~2 kDa, cysteine-rich peptides (CRPs) (described in paper III), were named St-CRP-1 and St-CRP-2 and contained 18-19 amino acids. The St-CRPs shared a disulfide connectivity pattern with alpha-defensins, had a neutral net charge, moderate antibacterial activity and showed no cytotoxicity. In addition, the introduction section provides background information on topics related to the thematic of the articles. This includes an introduction to bacteria, antibiotics and antibiotic resistance, AMPs, ascidians, and the marine environment

Antimicrobial Activity of Securamines From the Bryozoan Securiflustra securifrons

Natural products and their derivatives have served as powerful therapeutics against pathogenic microorganisms and are the mainstay of our currently available treatment options to combat infections. As part of our ongoing search for antimicrobial natural products from marine organisms, one fraction prepared from the Arctic marine bryozoan Securiflustra securifrons was found to be active against the human pathogenic bacterium Streptococcus agalactiae (gr. B). Chemical investigation of the fraction revealed that it contained several variants of the highly modified secondary metabolites known as securamines. The securamines are alkaloids sharing a common isoprene-histamine-tryptamine backbone. In this study, we describe the antimicrobial activities of securamine C, E, and H – J (4, 5, and 1-3) and the attempt to deconvolute the mode of action of 1

Identification of New Purpuroine Analogues from the Arctic Echinodermata Pteraster militaris That Inhibit FLT3-ITD+ AML Cell Lines

Isolation of bioactive products from the marine environment is considered a very promising approach to identify new compounds that can be used for further drug development. In this work we have isolated three new compounds from the purpuroine family by mass-guided preparative HPLC; purpuroine K-M. These compounds where screened for antibacterial- and antifungal activity, antibiofilm formation and anti-cell proliferation activity. Additionally, apoptosis-, cell cycle-, kinase binding- and docking studies were performed to evaluate the mechanism-of-action. None of the compounds showed activity in antibacterial-, antibiofilm- or antifungal assays. However, one of the isolated compounds, purpuroine K, showed activity against two cell lines, MV-4-11 and MOLM-13, two AML cell lines both carrying the FTL3-ITD mutation. In MV-4-11 cells, purpuroine K was found to increase apoptosis and arrest cells cycle in G1/G0, which is a common feature of FLT3 inhibitors. Interactions between purpuroine K and the FLT3 wild type or FLT3 ITD mutant proteins could however not be elucidated in our kinase binding and docking studies. In conclusion, we have isolated three novel molecules, purpuroine K-M, one of which (purpuroine K) shows a potent activity against FLT3-ITD mutated AML cell lines, however, the molecular target(s) of purpuroine K still need to be further investigated

Identification of New Purpuroine Analogues from the Arctic Echinodermata Pteraster militaris That Inhibit FLT3-ITD+ AML Cell Lines

Isolation of bioactive products from the marine environment is considered a very promising approach to identify new compounds that can be used for further drug development. In this work we have isolated three new compounds from the purpuroine family by mass-guided preparative HPLC; purpuroine K-M. These compounds where screened for antibacterial- and antifungal activity, antibiofilm formation and anti-cell proliferation activity. Additionally, apoptosis-, cell cycle-, kinase binding- and docking studies were performed to evaluate the mechanism-of-action. None of the compounds showed activity in antibacterial-, antibiofilm- or antifungal assays. However, one of the isolated compounds, purpuroine K, showed activity against two cell lines, MV-4-11 and MOLM-13, two AML cell lines both carrying the FTL3-ITD mutation. In MV-4-11 cells, purpuroine K was found to increase apoptosis and arrest cells cycle in G1/G0, which is a common feature of FLT3 inhibitors. Interactions between purpuroine K and the FLT3 wild type or FLT3 ITD mutant proteins could however not be elucidated in our kinase binding and docking studies. In conclusion, we have isolated three novel molecules, purpuroine K-M, one of which (purpuroine K) shows a potent activity against FLT3-ITD mutated AML cell lines, however, the molecular target(s) of purpuroine K still need to be further investigated

Antimicrobial peptides from the Arctic ascidian Synoicum turgens

The rise in frequency of antibiotic resistant pathogenic bacteria makes the need for new treatment options for previously curable bacterial infections ever more important. In the process of discovering and developing antibacterial agents, one powerful approach has been borrowing wisdom from nature. Antimicrobial peptides (AMPs) are critical components of the innate immune systems found in almost all eukaryotic life forms. Their mode of action includes disruption of the bacterial membrane and to trigger supporting immune responses. Due to these properties, AMPs are considered promising lead structures that can be further developed into commercially available antibiotics to treat or prevent human diseases caused by bacteria. The work conducted in this thesis aims to discover and characterize novel antimicrobial peptides from the Arctic marine ascidian Synoicum turgens by using a marine bioprospecting approach. This includes collection, extraction and fractionation of biomass, antibacterial bioactivity testing and AMP isolation followed by chemical and biological characterization. For one isolated peptide class, truncated versions were prepared, aiming to produce shorter, linear variants with retained antimicrobial activity. In paper I, turgencin A and B and their oxidized derivatives were discovered through bioassay-guided purification. These peptides had an unusual disulfide connectivity, rarely seen in marine AMPs. Both turgencin A and B were potently active against all assayed bacterial strains. Membrane assays showed that the peptides cause bacterial membrane disruption within a few seconds. Turgencin A and B also displayed some cytotoxic activity against two human cell lines. Oxidation of the methionine present in both peptides decreased the bioactivities in all assays. Turgencin A, being the most potent AMP, was subject for sequence analysis and prediction of antimicrobial potential of different regions in paper II. Truncated and modified variants of turgencin A were synthetically produced to make smaller AMPs with the potential of being developed into antimicrobial drug leads. These 10-mer peptides, named StAMP-1–11, were made using an amino acid replacement strategy. Some of the Trp enriched peptides had similar bactericidal activity as the parent peptide turgencin A, and no cytotoxic activity against the mammalian cell lines. During turgencin isolation, a series of other smaller peptides were discovered in the same extracts presumably with antimicrobial activity. These isolated and characterized ~2 kDa, cysteine-rich peptides (CRPs) (described in paper III), were named St-CRP-1 and St-CRP-2 and contained 18-19 amino acids. The St-CRPs shared a disulfide connectivity pattern with alpha-defensins, had a neutral net charge, moderate antibacterial activity and showed no cytotoxicity. In addition, the introduction section provides background information on topics related to the thematic of the articles. This includes an introduction to bacteria, antibiotics and antibiotic resistance, AMPs, ascidians, and the marine environment

Antimicrobial activity of small synthetic peptides based on the marine peptide turgencin A: Prediction of antimicrobial peptide sequences in a natural peptide and strategy for optimization of potency

Turgencin A, a potent antimicrobial peptide isolated from the Arctic sea squirt Synoicum turgens, consists of 36 amino acid residues and three disulfide bridges, making it challenging to synthesize. The aim of the present study was to develop a truncated peptide with an antimicrobial drug lead potential based on turgencin A. The experiments consisted of: (1) sequence analysis and prediction of antimicrobial potential of truncated 10-mer sequences; (2) synthesis and antimicrobial screening of a lead peptide devoid of the cysteine residues; (3) optimization of in vitro antimicrobial activity of the lead peptide using an amino acid replacement strategy; and (4) screening the synthesized peptides for cytotoxic activities. In silico analysis of turgencin A using various prediction software indicated an internal, cationic 10-mer sequence to be putatively antimicrobial. The synthesized truncated lead peptide displayed weak antimicrobial activity. However, by following a systematic amino acid replacement strategy, a modified peptide was developed that retained the potency of the original peptide. The optimized peptide StAMP-9 displayed bactericidal activity, with minimal inhibitory concentrations of 7.8 µg/mL against Staphylococcus aureus and 3.9 µg/mL against Escherichia coli, and no cytotoxic effects against mammalian cells. Preliminary experiments indicate the bacterial membranes as immediate and primary targets