DOI: 10.1002/cbic.201300246 Novel Aeruginosin-865 from Nostoc sp. as a Potent Anti-inflammatory Agent Aleksandra Kapus ´cik, [a] Pavel Hrouzek,* [a, b] Marek Kuzma, [c] Simona Bµrtovµ, [c, d] Petr Novµk, [c, e] Jouni Jokela, [f] Maren Pflüger, [g] Andreas Eger, [g] Harald Hundsberger, [g] and Jir ˇí Kopecky ´ [a] Introduction Inflammatory processes underlie many human diseases, con- tribute to pathogenesis of rheumatic, cardiovascular and Alz- heimer diseases, and increase the risk of cancer. [1–4] The con- nection between inflammation, innate immunity and cancer is widely accepted, however molecular mechanisms mediating this relationship have been only partly investigated. [3] Because treatment of many types of cancer has reached an efficacy pla- teau, there is a constant demand for new anti-inflammatory drugs for use in prevention or as a combined therapy with chemotherapeutics and radiation. [5] Cyanobacteria have proven to be an interesting source of novel chemical compounds with not only a broad diversity of structures, but also unique bioactivity patterns, [6, 7] including anti-inflammatory compounds acting as selective inflammatory pathway inhibitors, [8] antioxidants and cyclooxygenase-2 inhibi- tors, [9] lipopolysaccharide antagonists, [10] antiproliferative agents, [11] and much more. [12] The most investigated group of cyanobacterial metabolites are small peptides synthesized on nonribosomal synthetic pathways; [6, 7, 13] these are classified into several families, according to their structural features, and include microcystins, anabaenopeptins, microginins, and aeru- ginosins. [6] Aeruginosins are a group of tetrapeptides containing at the N terminus a derivative of hydroxy-phenyl lactic acid (Hpla), a variable amino acid, 2-carboxy-6-hydroxyoctahydroindole (Choi), and an arginine derivative at the C terminus, synthe- sized by nonribosomal peptide synthetase/polyketide synthase (NRPS/PKS) hybrid enzymes. [14] Almost 30 aeruginosin variants of have been isolated, from Microcystis, Planktothrix (Oscillato- ria), and Nodularia strains. [6, 15, 16] However, taking into consider- ation possible structural modifications, including sulfatation, glycosylation, and halogenation, the number of various conge- ners is estimated to be more than 500. [17] Recently aeruginosins have been an attractive research topic because of their inhibi- tory activity against trypsin-like serine proteases, which are im- portant targets in the search for new anticoagulants. [18, 19] Sev- eral reports have described their chemical synthesis, [20] struc- ture–activity relationship (SAR), and optimization of particular Aeruginosin-865 (Aer-865), isolated from terrestrial cyanobacte- rium Nostoc sp. Lukes ˇovµ 30/93, is the first aeruginosin-type peptide containing both a fatty acid and a carbohydrate moiety, and is the first aeruginosin to be found in the genus Nostoc. Mass spectrometry, chemical and spectroscopic analysis as well as one- and two-dimensional NMR and chiral HPLC analysis of Marfey derivatives were applied to determine the peptidic sequence: d-Hpla, d-Leu, 5-OH-Choi, Agma, with hex- anoic and mannopyranosyl uronic acid moieties linked to Choi. We used an AlphaLISA assay to measure the levels of proin- flammatory mediators IL-8 and ICAM-1 in hTNF-a-stimulated HLMVECs. Aer-865 showed significant reduction of both: with EC 50 values of (3.5 Æ 1.5) mg mL À1 ((4.0 Æ 1.7) mm) and (50.0 Æ 13.4) mg mL À1 ((57.8 Æ 15.5) mm), respectively. Confocal laser scanning microscopy revealed that the anti-inflammatory effect of Aer-865 was directly associated with inhibition of NF- kB translocation to the nucleus. Moreover, Aer-865 did not show any cytotoxic effect. [a] A. Kapus´cik, Dr. P. Hrouzek, Dr. J. Kopecky ´ Department of Phototrophic Microorganisms–ALGATECH Institute of Microbiology, Academy of Science of the Czech Republic Opatovicky ´ mly ´n, 379 81 Tr ˇebon ˇ (Czech Republic) E-mail : hrouzekp@gmail.com [b] Dr. P. Hrouzek Institute of Chemistry, Faculty of Sciences, University of South Bohemia Branisˇovskµ 31, 370 01C ˇ eskØ Bude ˘jovice (Czech Republic) [c] Dr. M. Kuzma, S. Bµrtovµ, Dr. P. Novµk Laboratory of Molecular Structure Characterization Institute of Microbiology, Academy of Science of the Czech Republic Víden ˇskµ 1083, 142 20 Prague (Czech Republic) [d] S. Bµrtovµ Department of Analytical Chemistry, Institute of Chemical Technology Technickµ 5, 166 28 Prague–Dejvice (Czech Republic) [e] Dr. P. Novµk Department of Biochemistry, Faculty of Sciences, Charles University Hlavova 8, 12840 Prague (Czech Republic) [f] Dr. J. Jokela Department of Food and Environmental Sciences Division of Microbiology, University of Helsinki P.O. Box 56, Viikinkaari 9, 00014 Helsinki (Finland) [g] M. Pflüger, Dr. A. Eger, Dr. H. Hundsberger Department of Medical and Pharmaceutical Biotechnology University of Applied Sciences Piaristengasse 1, 3500 Krems (Austria) Supporting information for this article is available on the WWW under http ://dx.doi.org/10.1002/cbic.201300246. # 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim ChemBioChem 2013, 14, 2329 – 2337 2329 CHEMBIOCHEM FULL PAPERS