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

Abstract

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.

Keywords: antibiotics; dereplication; marine sponges; natural products; plant pathogen.

Figure 1

Sampling sides of sponge specimen. Samples were retrieved by SCUBA in a depth of 4–20 m below the surface.

Figure 2

Schematic overview of the SeaPEPR pipeline. In a first step, crude environmental extracts are subject of bioactivity assessment. At the same time, the chemical diversity of the entire set of samples is determined by cosine similarity calculation (“chemical fingerprints”). Prioritized samples are microfractionated to identify the causative agent responsible for the initially observed bioactivity. If desired, dereplicated compounds of interest can be selected for isolation.

Figure 3

Cosine similarity heatmap of all 76 extracts. Blue color indicates a high degree of similarity among compared extracts (see color key histogram). Flags in sidebar mark selected samples for microfractionation (black) and screening results (red = active, orange = weak activity, white = inactive) of the respective extract against the indicator strains (CALB = C. albicans, STRI = S. tritici, SAUR = S. aureus, PAER = P. aeruginosa, ECOL = E. coli).

Figure 4

Base peak chromatogram (BPC) of the extracts KOL_08, KOL_16, KOL_18, and ULU_13 obtained from different Agelas nakamurai organisms. Most intense peaks within the similar BPCs correspond to the agelasines groups (box, top right) and agelasidine A (box, top left).

Figure 5

Chemical structures of the dereplicated compounds responsible for the activity of the microfractionated samples.

Figure 6

Underwater pictures and isolated spicules of the Agelas nakamurai cf specimens (a) Sample KOL_8, (b) Sample KOL_16, (c) Sample KOL_18, and (d) Sample ULU_13. It can be seen that the specimens are thick encrusting orange sponges and the type of spicule is megascleres acanthostyle for all four samples. This suggested the assignment as Agelas nakamurai cf.