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Natural products have been used as drugs and drug leads since the dawn of modern medicine. Most of these natural products are secondary metabolites (SMs), which means that they are not products of the primary metabolism that is shared by most living organisms, but are produced by specialized metabolic pathways that are unique for smaller groups of organisms. Most SMs used in medicine today have been isolated from terrestrial plants, fungi, and microorganisms.
The marine environment, specifically the Arctic seas, are some of the least explored environments on the planet, with untapped potential for producers of bioactive SMs. Much of the current research on marine biodiversity focuses on tropical organisms, so some researchers are looking toward cooler regions in the search for novel bioactive compounds. One such research group is Marbio at UiT—the Arctic University of Norway in Tromsø, which is focused on high-throughput screening and marine natural products drug discovery. The Marbio analytical laboratory has identified the niche of studying bioactive natural products in cold-adapted organisms, including invertebrates and microorganisms such as microalgae, bacteria, and fungi.
Marine bioprospecting
Marbio scientists work with Marbank—a national marine biobank coordinating a network of marine collections—to provide national and international academia and industry with access to marine biodiversity, its associated data, and extractable products. As part of this collaboration, Marbio scientists focus 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 this search have the potential for numerous drug discovery applications, including antibacterial, anticancer, immunostimulants, anti-inflammatories, antioxidants, and diabetes treatment.
The researchers at Marbio explore the potential of bioactive natural products by isolating previously uncultured bacterial strains from marine sediments, some of which will produce novel SMs. The search for these SMs from natural sources can be very time-consuming, hindered by the rediscovery of known compounds. Eliminating these known compounds is known as dereplication, and saves important time and resources. The most common method of dereplication in natural product drug discovery uses liquid chromatography/mass spectrometry (LC/MS) for sensitive analysis of complex mixtures. When using high-resolution MS, the accurate mass of the compound can be used to calculate the elemental composition, which can then be used to search databases to identify known molecules. The laboratory conducts a prefractionation of the extracts into approximately eight fractions, which are tested for bioactivity. LC/MS is used to compare active and inactive fractions and select the bioactive ones.
However, this approach only recognizes compounds that are identical to those in the databases, so any that are similar but not identical to existing compounds will not be identified. To overcome this limitation, the laboratory uses ion mobility separation (IMS) quadrupole time-of-flight (QTof) MS to add information on MS/MS fragmentation in the dereplication process, as fragments will be characteristic for common structural features in a molecular class. These fragment data can be used to search MS fragment libraries, such as the Global Natural Products Social Molecular Networking (GNPS).1 With this method, the laboratory can see the relationship between molecules, which was previously difficult simply using the elemental composition. Fragment data enable the visualization of specific fragmentation patterns that are similar, even though the accurate mass of the target molecule is different.
Discovering new bioactive compounds
Isolating the compound is usually the bottleneck in the laboratory, as it is the most time-consuming step. Before IMS QTof MS was introduced to a laboratory, when the target compound was selected, there was less certainty that it was the right compound. This led to cases where a compound was isolated that was not of interest, because it turned out to be a trivial metabolite that did not bring any new information. Around 30–40% of the compounds at this time were redundant and turned out not to be of interest. Since using IMS QTof MS, the laboratory has not experienced the isolation of any trivial molecules, and is more certain about the compounds it is working with.
A recent study by the Marbio group included the cultivation of a newly isolated Arctic marine Pseudomonas sp. strain M10B774, which is affiliated with the P. fluorescence group.2 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 nonmalignant normal lung fibroblast cell line (MRC5).
The newly isolated Pseudomonas sp. strain was cultured in four different media, which influenced the production of bioactive compounds. Culture extracts were prefractionated and screened for anticancer and antibacterial properties, and ultra-performance liquid chromatography (UPLC) and IMS QTof MS were performed to analyze the fractions (ACQUITY UPLC I-Class and Vion IMS QTof, Waters, Milford, MA). 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. Rhamnolipids, primarily those produced from P. aeruginosa, have been widely studied, and are currently produced industrially from this species. However, due to the human pathogenicity of P. aeruginosa, an alternative source of rhamnolipids, such as from the P. fluorescence group, would be beneficial as a potential drug candidate. It is therefore important to elucidate which rhamnolipids the M10B744 strain produces.
The project demonstrated the use of MS/MS-based molecular networking as a dereplication strategy to identify known compounds, their analogs, and related compounds. Five mono-rhamnolipids were characterized for the first time from a bacterium within the P. fluorescence group, including one new mono-rhamnolipid.
Advanced analytical capabilities
Due to the variable concentrations of molecules within fractions and the complexity of samples, technology with high dynamic range is required at the Marbio laboratory. The high degree of specificity provided by combining UPLC with IMS QTof MS enables clearer and more comprehensive data and, when combined with the sophisticated elucidation and database searching capabilities of modern software, such as the Waters UNIFI platform, allows researchers to 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.
Beyond drug discovery
Once Marbio researchers have identified new bioactive compounds in the discovery phase, their partners in industry continue through with commercialization of the novel drug leads. For example, the Arctic University of Norway collaborates with the Lead Discovery Center (LDC) in Dortmund, Germany, a center for translational medicine for the Max Planck Institute for Medical Research. The Marbio facility is identifying between 30 and 50 compounds per year, all of which can be isolated and tested for bioactivity.3
The laboratory is continually enhancing its work with emerging technology. The natural products community is increasingly linking different types of data and conducting more work on metabolite profiles. The university is also applying for funding to isolate compounds using supercritical fluid chromatography (SFC), which offers improved analytical scale and uses fewer organic solvents, minimizing environmental impact.
For the full case study detailing the Arctic University of Norway’s work, please visit https://www.waters.com/waters/library.htm?cid=511436&lid=135014384&xcid=ext7571
References
- Wang, M.; Carver, J.J. et al. Sharing and community curation of mass spectrometry data with GNPS. Nat. Biotechnol. 2016, 34, 828–37.
- Kristoffersen, V.; Rämä T et al. Characterization of rhamnolipids produced by an arctic marine bacterium from the Pseudomonas fluorescence group. Marine Drugs 2018, 16, 163.
- Cutting-Edge Analytical Technology Advances Arctic Marine Bioprospecting;Waters Corp, Feb 2019.
Dr. Espen Hansen is a professor at The Norwegian College of Fishery Science; https://en.uit.no/om/enhet/forsiden?p_dimension_id=88166