The main research themes in the marine natural products group are the functions and applications of natural products, particularly those from marine organisms. This encompasses two broad areas, the use of bioactive marine natural products as potential pharmaceuticals and tools for biomedical research and the interactions of these metabolites with transition metals.
Speculation as to the true function of these metabolites in their biological sphere is key to making advances in both these research areas. The core techniques involved in this work are in the separation and structure determination of natural products. Work in the group involves organic synthesis where compound is needed for further biological testing or to determine its ecological/biological function.
Work with molecular biologists is aimed at accessing the natural products through expressing their biosynthetic genes in heterologous hosts. Theoretical methods are used where these will lead to further insight into the biological or catalytic activity of the natural products or their complexes.
- Marine Natural Products as Potential Pharmaceuticals and Tools for Biomedical Research
Natural products are traditionally the cornerstone of drug discovery. A current reawakening of interest in natural products as useful lead compounds in diseases with no defined aetiology (cf ‘chemical genetics’) is stimulating the study of uninvestigated groups of organisms for their biosynthetic capacity.
Studies performed at the National Cancer Institute in the USA have shown that marine macro- and micro- organisms represent a significant source of biologically active lead compounds. We are looking at the isolation of novel drug candidates from soft-bodied marine organisms as well as marine microorganisms.
The further development of compounds from invertebrates can hampered by the problems in obtaining sufficient compound for follow-up studies, which may be circumvented by the use of microorganisms which can be re-cultured.
Collaborations are in place with the University of the South Pacific, Fiji, The Australian Institute of Marine Science and the National Biodiversity Institute (INBio) in Costa Rica. The research on marine microorganisms is in collaboration with Prof Goodfellow at the University of Newcastle (marine bacteria).
A biological testing arrangements are in place with Prof Fred Valeriote at the Ford Cancer Center in Detroit, USA and the lab of Marc Diederich in Luxembourg to discover new NF-kB inhibitors and with Mike Ferguson and Daan van Aalten in Dundee to find novel treatments for tropical diseases.
As an outcome of one of these collaborations, compound 1, isolated from a Fijian sponge has an IC50 = 5 ng/mL against ovarian carcinoma. This result is being followed up at the Ford Cancer Center in Detroit, USA.
Together with Rod Scott at the Institute of Medical Sciences in Aberdeen, we have been working on the unusual sponge compounds 2, the 1,3-alkyl pyridinium salt oligomers. We have discovered that these naturally occurring oligomers are able to form pores in cell membranes.
More recent work has shown that the larger size class (25 KDa) is able to reversibly form pores through which we have been able to pass the gene for GFP, to give viable transfected cells, which express the protein. In addition we have been able to use this method in vivo in rats to place exogenous proteins in brain cells. Patent protection has been applied for and we are currently seeking funding to commercialise this technology. We are synthesising regular oligomers and fluorescently labelled ones for mechanistic studies.
- Marine Bioinorganic Chemistry
We have been investigating the metal complexation abilities of metabolites of the Indo-Pacific ascidian (seasquirt) Lissoclinum patella. These are thiazole, thiazoline and oxazoline containing cyclic hepta and octapeptides (eg patellamide C 3). We discovered that patellamide C (3), binds selectively to Cu2+ in the presence of Zn2+, and does not bind to the other biologically important metal ions, Mg2+, Ca2+, Ni2+ and Co2+. In addition we have shown that Cu2+ can displace Zn2+ already bound to patellamide C.
The system is therefore selective for Cu2+ over these other divalent cations, which suggests possible biological functions in nature. The unit responsible for binding to copper in patellamide C is made of three nitrogen atoms in the thiazole, amide and oxazoline functionalities (marked T, A, O in 3).
We have attempted chemical approaches to compound 4 where F is a fluorescent group to generate fluorescent Cu(II) chemosensors, and measurements are being carried out on half molecules with the binding motif and a fluorescent group. We are also interested in investigating the use of other metal complexing compounds from ascidians as metal sensors, and ascididemin, a pyridoacridine alkaloid is proving very interesting in this respect.
The patellamides (3) are able to complex to two Cu2+ and these are 3.6 - 4.5Å apart which is similar to type III dimeric copper centres in proteins, which often function as oxygenases (Scheme 1 (5)) in biological systems. This distance suggests that a (m-h2:h2-O2) or (m-1,2-O2) bridge might be possible between the two coppers in the dicopper complex of 3. A suspicion that it might act as a catalase (Scheme 1 (3)) was confirmed by the ability of a low concentration of 3/2Cu2+ to decompose H2O2 to H2O and O2 with a maximum Kcat = 105 - 106 which is close to that of naturally occurring catalases.
Catalases are common in primitive photosynthetic systems to remove H2O2 which is an undesired intermediate in the course of photosynthetic water oxidation. Lissoclinum patella contains a primitive prokaryotic photosynthetic symbiont, Prochloron, thus suggesting a biological function for the catalase activity. Catalases often function as peroxidases (Scheme 1, (1) and (2)), and we will explore this possibility. In addition, preliminary electronic structure calculations with the Cu…Cu distance set to 4.5 Å appear to rule out dicopper cores (m-h2:h2-O2) and (m-O)2, but are consistent with the (m-1,2-O2) core. Copper containing enzymes often act as oxidases (Scheme 1 (4)) and this is another possibility we will investigate.
Scheme 1: Examples of reactions of peroxidases, catalases, oxidases and oxygenases
The patellamides may be part of an unrecognised family of catalytic species, the ‘catalytic natural products’, consisting of metal-binding non-ribosomal modified cyclic peptides. These low molecular weight compounds (<2 kDa) are highly conformationally constrained, and present the metal with an ideal coordination environment for it to become equivalent to an active site in a metalloprotein.
We feel that this may be a more general phenomenon that has gone unrecognised before now, and that there may be a large number of catalytic natural products to be discovered with diverse functions and potential uses.
- Molecular approaches to access marine natural products
We are collaborating with Prof Feldmann in Aberdeen, Dr Long at the London School of Pharmacy and Drs Battershill and Dunlap at the Australian Institute of Marine Science to investigate these unusual catalytically active complexes. We have collected ascidians (seasquirts) which hyperaccumulate transition metals and will isolate their DNA.
We will then capitalise on our recent success in achieving heterologous expression of an ascidian metabolite and attempt heterologous expression of the other ascidian DNA in suitable hosts so that all biosynthetic pathways can be expressed, as natural product expression in natural populations can be very variable. We will then seek to find the metabolites of interest using high-throughput metal binding assays.
Positive hits will be grown in bulk culture and isolated and identified using spectroscopic methods.
The main importance of this work is the discovery of small molecule systems with metalloenzyme-like properties. One important activity might be chiral oxidation of substrates, including those, which are hard to oxidise by other means. The catalysts are likely to give large rate accelerations with high turnover numbers. They should work at room temperature, and some of them will work in water.
In addition these complexes might be more robust than enzymes. In Nature, it is possible that some of these compounds are ‘isolated active sites’. The disposition of the ligating atoms and coordination sphere of the metal is the same as a much larger enzyme, but it may be necessary for it to be immobilised in a membrane for it to function efficiently. This programme will provide a sustainable means to produce and investigate these compounds.
In future, altering the biosynthetic genes by mutation or molecular biology methods may enable the ‘tuning’ or altering the catalytic activity. In addition, these metal complexing agents, and these may prove useful as treatments for diseases of metal accumulation (eg Wilson’s disease), and even in the treatment of Alzheimer’s disease in which aberrant Cu(II) or Zn(II) homeostasis is hypothesised to be a causative factor in the disease. Thiol-disulfide exchange reactions play a key role in many metabolic processes.
We will investigate compounds containing disulphide bridges which may act as ligands to complex metal ions for the development of new classes of antimicrobial drugs and anticancer agents.
- Biosynthesis and Exploitation of Marine-Derived Post-Translationally Modified Ribosomal Peptides
Modified peptides display a wealth of biological activity and most are constructed via NRPS pathways. An emerging group of metabolites is the post-translationally modified ribosomal peptides. These metabolites are formed from a ribosomally synthesised prepeptide by a complex of monofunctional peptides. Knowledge of the metabolite’s chemical structure will therefore enable the prediction of the gene sequence from which it is derived. The biosynthetic rules towards these metabolites are not yet clearly understood.
The most complex examples of this class are the patellamides, originally isolated from the seasquirt Lissoclinum patella, but now confirmed to be synthesised by its symbiont, Prochloron didemni. Many seasquirts and sponges produce related peptides that are often attributed to the symbiont. The biosynthesis of many of these bioactive compounds may also be via a ribosomal pathway.
The main aim is to use a combination of chemistry, chemoinformatics, molecular biology and bioinformatics to explore these natural products. The discovery of novel biosynthetic pathways towards these bioactive modified peptides would be of interest academically, and will make excellent candidates for biosynthetic engineering. There are two strands to this work; (a) understanding the biosynthetic rules and (b) appreciation of the diversity of these type of compounds in the natural environment and gaining an understanding of how the pathways vary.
A large number of molecules have been described from marine sources that are likely to be synthesised by this pathway. We will reisolate these compounds, and isolate the symbiont and its genomic DNA. Following this, the design of degenerate primers for conserved domains in the gene cluster will allow the systematic discovery of novel compounds in this class. Detailed cataloguing of the types of sea squirt and the nature of their symbionts (16S rRNA determination) will enable relationships between these organisms to be understood.