Sustainable Low-Volume Analysis of Environmental Samples by Semi-Automated Prioritization of Extracts for Natural Product Research (SeaPEPR).

The discovery of novel natural products (NPs) that will serve as lead structures has to be an ongoing effort to fill the respective development pipelines. However, identification of NPs, which possess a potential for application in e.g., the pharma or agro sector, must be as cost effective and fast as possible. Furthermore, the amount of sample available for initial testing is usually very limited, not least because of the fact that the impact on the environment, i.e., the sampled biosystem, should be kept minimal. Here, our pipeline SeaPEPR is described, in which a primary bioactivity screening of crude extracts is combined with the analysis of their metabolic fingerprint. This enabled prioritization of samples for subsequent microfractionation and dereplication of the active compounds early in the workflow. As a case study, 76 marine sponge-derived extracts were screened against a microbial screening panel. Thereunder, human pathogenic bacteria (Escherichia coli ATCC35218 and Staphylococcus aureus ATCC33592) and yeast (Candida albicans FH2173), as well as the phytopathogenic fungus Septoria tritici MUCL45407. Overall, nine extracts revealed activity against at least one test organism. Metabolic fingerprinting enabled assigning four active extracts into one metabolic group; therefore, one representative was selected for subsequent microfractionation. Dereplication of the active fractions showed a new dibrominated aplysinopsin and a hypothetical chromazonarol stereoisomer derivative. Furthermore, inhibitory activity against the common plant pest Septoria tritici was discovered for NPs of marine origin.

Selection of sponge-associated bacteria with high potential for the production of antibacterial compounds.

The potential of sponge-associated bacteria for the biosynthesis of natural products with antibacterial activity was evaluated. In a preliminary screening 108 of 835 axenic isolates showed antibacterial activity. Active isolates were identified by 16S rRNA gene sequencing and selection of the most promising strains was done in a championship like approach, which can be done in every lab and field station without expensive equipment. In a competition assay, strains that inhibited most of the other strains were selected. In a second round, the strongest competitors from each host sponge competed against each other. To rule out that the best competitors selected in that way represent similar strains with the same metabolic profile, BOX PCR experiments were performed, and extracts of these strains were analysed using metabolic fingerprinting. This proved that the strains are different and have various metabolic profiles, even though belonging to the same genus, i.e. Bacillus. Furthermore, it was shown that co-culture experiments triggered the production of compounds with antibiotic activity, i.e. surfactins and macrolactin A. Since many members of the genus Bacillus possess the genetic equipment for the biosynthesis of these compounds, a potential synergism was analysed, showing synergistic effects between C14-surfactin and macrolactin A against methicillin-resistant Staphylococcus aureus (MRSA).

A New Sesquiterpenoid Aminoquinone from an Indonesian Marine Sponge.

Sponges are a well-known bioresource for bioactive compounds. In this study, antibacterial activity-guided fractionation of the extract from an Indonesian marine Dactylospongia elegans sponge led to the discovery of four merosesquiterpenoids, namely, a new sesquiterpenoid aminoquinone nakijiquinone V (1), along with illimaquinone (2), smenospongine (3), and dyctioceratine C (4). The structure of compound 1 was elucidated by 1D and 2D NMR as well as by LC-HRESIMS data analysis. Compounds 2⁻4 showed moderate to low antimicrobial activity against Bacillus megaterium DSM32 with a minimum inhibitory concentration (MIC) of 32 µg/mL, 32 µg/mL, and 64 µg/mL, respectively. Furthermore, compounds 2 and 3 both inhibited Micrococcus luteus ATCC 4698 with a MIC of 32 µg/mL. In conclusion, the isolated merosesquiterpenoids, which are known for their cytotoxic effects, showed antibacterial activity and prompt future structure activity relationship (SAR) studies concerning the various bioactivities observed for this group of natural products.

Sesterterpene glycinyl-lactams: a new class of glycine receptor modulator from Australian marine sponges of the genus Psammocinia.

Bioassay guided fractionation of three southern Australian marine sponges of the genus Psammocinia, selected for their ability to modulate glycine-gated chloride channel receptors (GlyRs), yielded the rare marine sesterterpenes (-)-ircinianin (1) and (-)-ircinianin sulfate (2), along with the new biosynthetically related metabolites (-)-ircinianin lactam A (3), (-)-ircinianin lactam A sulfate (4), (-)-oxoircinianin (5), (-)-oxoircinianin lactam A (6) and (-)-ircinianin lactone A (7). Acetylation of 1 returned (-)-ircinianin acetate (8). Whole cell patch-clamp electrophysiology on 1-8 established 3 as an exceptionally potent and selective α3 GlyR potentiator, and 6 as a selective α1 GlyR potentiator. The discovery and characterization of sesterterpenes 1-8, and in particular the glycinyl-lactams 3 and 6, provide valuable new insights into GlyR pharmacology. These insights have the potential to inform and inspire the development of new molecular tools to probe GlyR distribution and function, and therapeutics to treat a wide array of GlyR mediated diseases and disorders.

Australian marine sponge alkaloids as a new class of glycine-gated chloride channel receptor modulator.

Chemical analysis of a specimen of the sponge Ianthella cf. flabelliformis returned two new sesquiterpene glycinyl lactams, ianthellalactams A (1) and B (2), the known sponge sesquiterpene dictyodendrillin (3) and its ethanolysis artifact ethyl dictyodendrillin (4), and five known sponge indole alkaloids, aplysinopsin (5), 8E-3'-deimino-3'-oxoaplysinopsin (6), 8Z-3'-deimino-3'-oxoaplysinopsin (7), dihydroaplysinopsin (8) and tubastrindole B (9). The equilibrated mixture 6/7 exhibited glycine-gated chloride channel receptor (GlyR) antagonist activity with a bias towards α3 over α1 GlyR, while tubastrindole B (9) exhibited a bias towards α1 over α3 GlyR. At low- to sub-micromolar concentrations, 9 was also a selective potentiator of α1 GlyR, with no effect on α3 GlyR-a pharmacology that could prove useful in the treatment of movement disorders such as spasticity and hyperekplexia. Our investigations into the GlyR modulatory properties of 1-9 were further supported by the synthesis of a number of structurally related indole alkaloids.

Ircinialactams: subunit-selective glycine receptor modulators from Australian sponges of the family Irciniidae.

Screening an extract library of >2500 southern Australian and Antarctic marine invertebrates and algae for modulators of glycine receptor (GlyR) chloride channels identified three Irciniidae sponges that yielded new examples of a rare class of glycinyl lactam sesterterpene, ircinialactam A, 8-hydroxyircinialactam A, 8-hydroxyircinialactam B, ircinialactam C, ent-ircinialactam C and ircinialactam D. Structure-activity relationship (SAR) investigations revealed a new pharmacophore with potent and subunit selective modulatory properties against alpha1 and alpha3 GlyR isoforms. Such GlyR modulators have potential application as pharmacological tools, and as leads for the development of GlyR targeting therapeutics to treat chronic inflammatory pain, epilepsy, spasticity and hyperekplexia.