Dr Espen Hansen discusses marine bioprospecting with advanced UPLC and MS technology
The majority of drugs on the market are derived from nature, but still there is a wide range of entirely unknown sources of compounds to develop novel drugs, most of which are untapped but could lead to treatments for a range of diseases and infections. In the ongoing search for new, more effective medicines, researchers are looking towards some of the least explored environments to discover organisms with the potential to provide unique bioactive natural products.
Current Research - Marine Biodiversity
One such environment is the Arctic, specifically the sea. The majority of current research on marine biodiversity concentrates on tropical organisms, so researchers at the Marbio analytical laboratory, Arctic University of Norway, Tromsø, are looking towards cold-adapted organisms for their work on high-throughput screening and natural marine products discovery. These organisms include invertebrates and microorganisms such as microalgae, bacteria and fungi.
What Is Marine Bioprospecting?
Marbio scientists focus their research on marine bioprospecting – the systematic search for interesting and unique genes, molecules, and organisms from the marine environment with features that could be useful to society and/or have potential for commercial development. The natural products resulting from bioprospecting are useful in many drug discovery applications, such as antibacterial, anticancer, immunostimulants, anti-inflammatories, antioxidants and diabetes treatment.
The laboratory isolates previously uncultured bacterial strains from marine sediments, some of which will produce novel secondary metabolites that could be translated into drugs. The search for these secondary metabolites from natural sources can be laborious in part because of rediscovery of known compounds, but the Marbio group saves vital time and resources by eliminating these compounds by a process called “dereplication”. Liquid chromatography mass spectrometry (LC-MS) is most commonly used for dereplication in natural product drug discovery, as this combination is both sensitive and well suited for analysing complex mixtures. The accurate mass of the compound can be used to calculate the elemental composition via high resolution MS, and then used to search databases such as MarinLit, Dictionary of Natural Products, and SciFinder, to identify unknown molecules.
Assessing Bioactivity
The laboratory pre-fractionates the extracts into approximately eight fractions, which are tested for bioactivity, before using LC-MS to differentiate and select bioactive fractions from inactive fractions. However, any similar but non-identical compounds to those existing in the database will not be identified, so the laboratory uses ion mobility separation (IMS) quadrupole time-of-flight (QTof) MS to add information on MS/MS fragmentation in the dereplication process. These fragment data can be used to search MS fragment libraries, such as the Global Natural Products Social molecular networking (GNPS), to visualise specific fragmentation patterns that are similar, even though the accurate mass of the target molecule is difficult.
Isolating the compound is the most time-consuming step of bioprospecting studies, and sometimes a compound is isolated that ultimately is a trivial metabolite that contributes no new information. Since IMS QTof MS technology was introduced to the Marbio laboratory, no trivial molecules have been isolated, and researchers can be more certain about the compounds they are working with.
Characterisation of Rhamnolipids
Recent work by the Marbio group shows the cultivation of a newly isolated arctic marine Pseudomonas sp. strain M10B774, which is affiliated with the P. fluorescence group. Fractions of the culture extracts were screened for antibacterial activity against the pathogenic bacteria Staphylococcus aureus, Enterococcus faecalis, Streptococcus agalactiae, Escherichia coli, and Pseudomonas aeruginosa in a growth inhibition assay. Cytotoxic activity of the fractions was also evaluated against three cancer cell lines, human melanoma (A2058), human breast carcinoma (MCF7), and human colon carcinoma (HT29), as well as the non-malignant normal lung fibroblast cell line (MRC5).
Pseudomonas sp. was cultivated in four different media to activate biosynthetic pathways leading to the production of antibacterial and anticancer compounds, and culture extracts were pre-fractionated and screened for anticancer and antibacterial properties. Active fractions were dereplicated using molecular networking based on MS/MS fragmentation data, indicating the presence of a cluster of related rhamnolipids – compounds known to have antibacterial and cytotoxic activities. Six compounds were isolated using high performance liquid chromatography (HPLC) and mass-guided fractionation, and by interpreting the data from nuclear magnetic resonance (NMR) and high-resolution MS/MS analysis.
The project demonstrated the use of MS/MS-based molecular networking as a dereplication strategy to identify known compounds, their analogs and related compounds. For the first time, five mono-rhamnolipids from a bacterium within the P. fluorescence group were characterised, including one new mono-rhamnolipid, as well as the lipid moiety of one of the rhamnolipids. In addition, one fraction from three of the four growth conditions showed inhibitory activity towards bacteria and cancer cells.
Instruments with high dynamic range are vital to the work at Marbio laboratory, due to the variable concentrations of molecules within fractions and the sample complexity. The sensitivity of IMS QTof MS is also required for users to distinguish between small and large peaks, to identify unknown molecules. The high degree of specificity provided by combining UPLC with IMS QTof MS also allows clearer and more comprehensive data and, when joined with modern software solutions for elucidation and database searching, researchers can mine more information to find new compounds faster. Such software platforms significantly reduce the data processing bottleneck by providing rapid access to results through efficient data acquisition and processing steps.
Future Directions
Collaboration is vital for the Arctic University of Norway to progress newly discovered bioactive compounds through the pharmaceutical development pipeline to commercialisation. The university’s industry partners, for example those at the Lead Discovery Center (LDC) at the Max Planck Institute for Medical Research, Germany, facilitate the development of a selection of the 30-50 compounds isolated by the Marbio laboratory per year.
To continue discovering new drug candidates, the laboratory is continually improving its methods and workflows with newly emerging technology. The natural products community is increasingly linking different types of data and conducting more work on metabolite profiles. The university is also minimising its environmental impact by applying for funding to isolate compounds using supercritical fluid chromatography (SFC), which offers improved analytical scale and uses less organic solvents.
Dr Espen Hansen is a professor in marine biotechnology at Marbio
