eXploring the BIOactive PEPtide Space of arctic marine invertebrates

The main aim of the XBioPepS project has been to identify, isolate and explore antimicrobial peptides (AMPs) and pain inhibitory peptides from Artic and Sub-Arctic marine invertebrates and to identify potential lead structures of pharmaceutical interest. The main focus have been towards deliveries on i) collecting marine organisms, ii) make peptides and nucleic acid extracts, iii) screen for antimicrobial activity, iv) perform more extensive studies of structure (SAR) and mechanisms of actions on selected peptides, v) deep sequencing and bioinformatics, vi) develop novel assays, vii) evaluate the commercial potential. The main motivation is the spreading of antibiotic resistance in bacteria against commercial antibiotics. Several human pathogenic bacteria are now resistant to any commercial antibiotic and we need novel compounds with new mechanisms of actions. The marine environment is particular interesting as a source for novel antibiotics for several reasons. During the last 34 years, the nature has been the source of 69% of novel drugs, but only 1% of these have been from the marine environment. For millions of years, the marine organism have evolved and adjusted perfectly to the marine bacteria. The repertoire of bioactive substances in marine organisms is varied. For many years, UiT have identified and explored bioactives like AMPs and their mechanisms of actions from different marine invertebrates. In addition to focus on Echinoderms and Crustacean, also Cnidaria and Mollusca are analysed. A bioassay guided purification strategy from extracts was used to identify novel antimicrobials and peptide toxins acting on neuroreceptors/ion channels. Several potent antimicrobial peptides in the µM range was isolated from extracts of the hemocytes of the red sea urchin, Echinus esculentus. These peptides resembled the already known strongylocins and centrocins from the green sea urchin (Strongylocentrotus droebachiensis). The peptides were posttranslational modified with brominated trypsins. The centrocins consisted of two peptides (dimers) connected by a disulphide bridge. The pharmacophores (the part responsible for the activity) resided in the heavy chain of the antimicrobial peptide. Structure activity relationship studies (SAR studies) and peptidomic studies revealed that the heavy chain of the EeCentrocin 1 could be shortened from the original 30 amino acids down to less than half of the original amount and still be potent. Work is still ongoing to optimize lead peptides by constructing truncated peptides based on the N-terminal pharmacophore and replace residues leading to peptides that are more active. This information can be the basis for designing novel peptidomic molecules. These are not real peptides, but contain pharmacophores and functional parts of the original natural or truncated peptides but is not so easily broken down by enzymes (proteases). Solving structures of natural peptides is challenging if you have small amounts and they contain several cysteines that form disulphide bridges. By using high resolution tandem mass spectrometry it was possible to detect the structure of novel peptides from the red king crab (Paralithodes camtschaticus) that al contained 8 cysteines. An example of peptidomics is the design and synthesis of marine natural products mimics (MNPMs) and their activity and mechanisms that is reported in this project. The work and design of novel chemical structures and bioactivity testing is still ongoing. Bioactive peptides and the genes were also isolated and characterized from sea anemones, Urticina eques and Metridium senile. One peptide (Ueg 12-1) showed a dual activity. In addition, of being antimicrobial active, it also showed potentiating activity on the TRPA 1 ion channel that is a known pain receptor. Another peptide (Ms 9a-1) decreased the pain and may thus be useful as an anti-inflammatory compound. The genes of the antimicrobials were identified and subjected for bioinformatic analysis. However, we were not able by deep sequencing (transcriptomics) of these animals to identify the peptides. In the future, the animals has to be stimulated before sampling and different samples taken simultaneously. Conventional antimicrobial screening based on growth inhibiting assays take one or two days to perform and we focused to make novel biosensor assays that is quicker (seconds, minutes or hours) and can be used to identify different mechanism of antimicrobial peptides at an early time point. The use of these assays in a screening process on unfractionated extracts showed promising results. In conclusion, the main aim was achieved. We have brought basic knowledge of peptides from marine organism and have used novel structure analysis. We have shown that we can design new active compounds with potent antimicrobial activity based on information from natural peptides. We have a patent application and we can soon offer new MOA assays.

The decreasing number of approved drugs produced by the pharmaceutical industry demands alternative approaches to increase pharmaceutical R&D productivity. This situation has contributed to a revival of interest in peptides as potential drug candidates. N ew synthetic strategies for limiting metabolism and alternative routes of administration have emerged in recent years and resulted in an increasing number of peptide-based drugs that are now being marketed. The emergence and spread of antimicrobial resist ance in human pathogenic bacteria has fuelled the need for new antimicrobial compounds with new mechanisms of action. There is also a critical need for novel therapeutics for pain management. Marine invertebrates have proven to be a vast resource for dis covering novel bioactive peptides, having either antimicrobial or pain-killing effects. These facts introduce bioactive peptides from marine invertebrates as a new choice for obtaining promising drug lead structures. The aim of the present project is to c haracterise antimicrobial and pain inhibitory peptides from marine invertebrates and to identify potential lead structures of pharmaceutical interest. The research will build on results from ongoing project activities at the University of Tromsø (UiT). A successful drug discovery relies on the coordinated multi-disciplinary effort of highly qualified researchers with special skills in different disciplines. Four different research groups at the University of Tromsø will join forces together with research partners at (NILU, in Stavanger, Germany, Finland and Russia. The national marine biobank (Marbank) and the screening platform (Marbio) take part in the project and optimization of extraction will be done to collect and characterise bioactive peptides of the animals. Our laboratories and collaborating local, national and international partners have the relevant technological and scientific expertise, as well as the motivation, to fulfil all the project aims.