Isolation and Characterization of Antiinfective Metabolites from Cyanobacteria

Abstract

Cyanobacteria, whilst initially having been neglected by natural product research for a long time, nowadays are recognized as a prolific source of structurally diverse and pharmacologically active natural products, like protease inhibitors. The present study is embedded in the bilateral project "Accessing Novel Bacterial Producers from Biodiversityrich Habitats in Indonesia" (AnoBIn) and aims to identify and isolate antiinfective metabolites from cyanobacteria, particularly inhibitors of the trypanosomal cystein protease rhodesain. Therefore, we screened a cyanobacteria extract collection generated as part of the ANoBIn project, as well as a library of 572 cyanobacteria extracts, kindly provided by Cyano Biotech GmbH, Berlin, against the trypanosomal cystein protease rhodesain in cooperation with Prof. Dr. Tanja Schirmeister (Johannes Gutenberg University Mainz). In order to constitute a cyanobacteria strain collection, various aquatic and terrestric ecosystems in Germany and Indonesia were sampled, followed by the isolation, characterization and biobanking of 147 cyanobacterial strains. The sampling in Germany led to the isolation of 55 strains from which 30.9% were belonging to the Chroococcales, 65.5% to the Oscillatoriales and 3.6% to the Nostocales. From 92 cyanobacterial strains isolated from Indonesian samples, 17.4% were allocated to the Chroococcales, 38.0% to the Oscillatoriales, 34.8% to the Nostocales and 9.8% to the Stigonematales. About 60 biomass extracts of the newly established strain collection were tested against three different reporter strains for activity on Quorum Sensing (QS) systems, antimicrobial activity against Bacillus subtilis and Staphylococcus aureus, and against the cystein protease rhodesain. The Cathepsin L like cysteine protease rhodesain plays an essential physiological role in the metabolism of the parasite Trypanosoma brucei. A human infection with Trypanosoma brucei is known as human African trypanosomiasis (HAT). Because of its important role, rhodesain is regarded as a promising target for the development of new medications. Of 52 tested extracts, 11.5% showed more than 50% inhibition of rhodesain at 0.1 mg/mL. Although 27 of the tested strains belonged to Oscillatoriales (III), only 7.4% of all tested Oscillatoriales strains showed pronounced inhibitory activity, while 15.4% of the Chroococcales (I), 12.5% of the Nostocales (IV) and 25% of the Stigonematales (V) strains showed more than 50% inhibition. Five of the screened extracts, revealing interesting bioactivity combined with a promising chemical profile, were selected for more intensive evaluation. The obtained HPLC-DAD and HPLC-MS data were assessed, and several known and new intriguing, possibly bioactive compounds could be identified.

Additionally, a library of 572 cyanobacteria extracts (kindly provided by Cyano Biotech GmbH, Berlin, Germany), was screened against the trypanosomal cystein protease rhodesain. The screening revealed 2.7% of the biomass extracts showing an inhibitory activity of more than 70% at 0.1 mg/mL, whilst 14.8% of the medium extracts featured an inhibition higher than 70% at 0.2 mg/mL. The biomass extract of a Nostoc sp. strain showed 98% inhibition at 0.1 mg/mL. Processing of this promising extract resulted in the isolation of 9 compounds, from which 5 showed pronounced inhibitory activity. Due to low yields, solely one compound was subjected to nuclear magnetic resonance (NMR) experiments, which successfully led to partial elucidation of this compound with a mass of [M+H]+ m/z 604.24 and a suggested molecular formula C24H38N5O13, comprising a sugar backbone (triose) and a nitrogen-rich side chain. With an inhibition of more than 90%, a distinct Nostoc sp. medium extract revealed conspicuous activity. We identified the known compound nostotrebin 6 and several novel biosynthetically related structures as active substances. Nostotrebin 6 is the only cyanobacterial cyclopentenedione (CDP) known to date and was already isolated from a Nostoc sp. biomass extract. It has been found to be active in a broad variety of assays, possibly convicting it as pan-assay interference compound (PAIN). The additionally isolated derivatives of nostrotrebin 6 were found to be oligomeric molecules, composed of two core monomeric structures, a trisubstituted CPD or a trisubstituted unsaturated δ-lactone. A comparative bioactivity testing revealed possibly unspecific rhodesain inhibition, antimicrobial activity and cytotoxicity, depending on the amount of free phenolic hydroxyl groups per molecule. To our knowledge, this work is the first to consider cyanobacteria as a source for rhodesain inhibitors. Within this study, we found 2% of a self-established biomass extract collection to show rhodesain inhibiting activity higher than 70% at 0.1 mg/mL. These results were in line with the screening of the commercial extract collection provided by the Cyano Biotech GmbH, where 2.7% of the biomass extracts showed an inhibition equal or higher than 70% at 0.1 mg/mL. Here, the section Nostocales displayed the major amount of inhibitory active strains with 55.6%. Until today, only few compounds from cyanobacteria cultivation media are known, but the present findings suggest that cyanobacteria, especially cyanobacterial medium extracts, provide a valuable source for protease inhibitors.