Discovery pipelines for marine resources: an ocean of opportunity for biotechnology?

Marine microbial diversity offers enormous potential for discovery of compounds of crucial importance in healthcare, food security and bioindustry. However, access to it has been hampered by the difficulty of accessing and growing the organisms for study. The discovery and exploitation of marine bioproducts for research and commercial development requires state-of-the-art technologies and innovative approaches. Technologies and approaches are advancing rapidly and keeping pace is expensive and time consuming. There is a pressing need for clear guidance that will allow researchers to operate in a way that enables the optimal return on their efforts whilst being fully compliant with the current regulatory framework. One major initiative launched to achieve this, has been the advent of European Research Infrastructures. Research Infrastructures (RI) and associated centres of excellence currently build harmonized multidisciplinary workflows that support academic and private sector users. The European Marine Biological Research Infrastructure Cluster (EMBRIC) has brought together six such RIs in a European project to promote the blue bio-economy. The overarching objective is to develop coherent chains of high-quality services for access to biological, analytical and data resources providing improvements in the throughput and efficiency of workflows for discovery of novel marine products. In order to test the efficiency of this prototype pipeline for discovery, 248 rarely-grown organisms were isolated and analysed, some extracts demonstrated interesting biochemical properties and are currently undergoing further analysis. EMBRIC has established an overarching and operational structure to facilitate the integration of the multidisciplinary value chains of services to access such resources whilst enabling critical mass to focus on problem resolution.

High-Throughput Fluorescence Polarization Assay to Identify Ligands Using Purified G Protein-Coupled Receptor.

The development of cell-free high-throughput (HT) methods to screen and select novel lead compounds remains one of the key challenges in G protein-coupled receptor (GPCR) drug discovery. Mutational approaches have allowed the stabilization of GPCRs in a purified and ligand-free state. The increased intramolecular stability overcomes two major drawbacks for usage in in vitro screening, the low receptor density on cells and the low stability in micelles. Here, an HT fluorescence polarization (FP) assay for the neurotensin receptor type 1 (NTS1) was developed. The assay operates in a 384-well format and is tolerant to DMSO. From a library screen of 1272 compounds, 12 (~1%) were identified as primary hits. These compounds were validated in orthogonal assay formats using surface plasmon resonance (SPR), which confirmed binding of seven compounds (0.6%). One of these compounds showed a clear preference for the orthosteric binding pocket with submicromolar affinity. A second compound revealed binding at a nonorthosteric binding region and showed specific biological activity on NTS1-expressing cells. A search of analogs led to further enhancement of affinity, but at the expense of activity. The identification of GPCR ligands in a cell-free assay should allow the expansion of GPCR pharmaceuticals with antagonistic or agonistic activity.

Biophysical characterization of E. coli TolC interaction with the known blocker hexaamminecobalt.

BACKGROUND: The tripartite efflux pump AcrAB-TolC in E. coli is involved in drug resistance by transporting antibiotics out of the cell. The outer membrane protein TolC can be blocked by various cations, including hexaamminecobalt, thereby TolC represents a potential target for reducing antimicrobial resistance as its blockage may improve efficacy of antibiotics. METHODS: We utilized single channel electrophysiology measurements for studying TolC conductance in the absence and presence of the known TolC blocker hexaamminecobalt. Association and dissociation constants of hexaamminecobalt were determined using surface plasmon resonance measurements. Minimum inhibitory concentration (MIC) assays in the absence and presence of antibiotics were carried out for investigating the antibacterial effect of hexaamminecobalt and its potential to reduce MICs. RESULTS: TolC gating in the absence of any ligand is voltage dependent and asymmetric at high applied voltages. Hexaamminecobalt binds to TolC with high affinity and kinetic data revealed fast association and dissociation rates. Despite potent binding to TolC, hexaamminecobalt does not possess an intrinsic antimicrobial activity against E. coli nor does it reduce MIC values of antibiotics erythromycin and fusidic acid. CONCLUSIONS: TolC opening can be effectively blocked by small molecules. More potent channel blockers are needed in order to investigate the eligibility of TolC as drug target. GENERAL SIGNIFICANCE: TolC, a potentially interesting pharmaceutical target can be addressed by small molecules, blocking the channel. Biophysical characterization of the binding processes will support future identification and optimisation of more potent TolC blockers in order to validate TolC as a pharmaceutical target.

Feasibility study using surface-enhanced Raman spectroscopy for the quantitative detection of tyrosine and serine phosphorylation.

We investigate the feasibility of colloid-based surface enhanced Raman scattering (SERS) as a highly sensitive technique for detecting peptide phosphorylation at serine and tyrosine residues. Using the recently reported drop-coating deposition Raman method we validate our SERS spectra against normal Raman spectra that would otherwise be unobtainable at such low concentrations. Compared with existing techniques for quantifying peptide phosphorylation, such as high-performance liquid chromatography (HPLC), the short scanning and processing time associated with SERS makes it an attractive alternative for near-real-time measurement at sub micro-molar concentrations. Following pre-processing by Savistky-Golay second derivative (SGSD), the degree of phosphorylation of synthetic peptides is determined using multivariate spectral classification, interval partial least squares (iPLS). Furthermore, our results show that the technique is robust to interference from complex proteins and other phosphorylated compounds present at concentrations typically found in a screening assay.

The analysis of intermolecular interactions in concentrated hyaluronan solutions suggest no evidence for chain-chain association.

Confocal fluorescence recovery after photobleaching (confocal-FRAP) was used to examine the influence of electrolytes (NaCl, KCl, MgCl(2), MnCl(2) and CaCl(2)) on the network and hydrodynamic properties of fluoresceinamine-labelled hyaluronan (FA-HA) at concentrations up to 10 mg/ml. Self and tracer lateral diffusion coefficients showed that in Ca(2+) and Mn(2+), FA-HA (830 kDa) was more compact than in Mg(2+), Na(+) or K(+). These results were correlated with changes in the hydrodynamic radius of HA, determined by multi-angle laser-light-scattering analysis in dilute solution, which was smaller in CaCl(2) (36 nm) than in NaCl (43 nm). The permeability of more concentrated solutions of HA (<10 mg/ml) to FITC-dextran tracers (2000 kDa) was higher in CaCl(2). The properties of HA in urea (up to 6 M) were investigated to test for hydrophobic interactions and also in ethanol/water (up to 62%, v/v). In both, there was reduced hydrodynamic size and increased permeability to FITC-dextran, suggesting increased chain flexibility, but it did not show the changes predicted if chain-chain association was disrupted by urea, or enhanced by ethanol. Oligosaccharides of HA (HA(20-26)) also had no effect on the self diffusion of high-molecular-mass FA-HA (830 kDa) solutions, or on dextran tracer diffusion, showing that there were no chain-chain interactions open to competition by short-chain segments. The results suggest that the effects of electrolytes and solvent are determined primarily by their effect on HA chain flexibility, with no evidence for association between chain segments contributing significantly to the major properties.

The molecular basis of the solution properties of hyaluronan investigated by confocal fluorescence recovery after photobleaching.

Hyaluronan (HA) is a highly hydrated polyanion, which is a network-forming and space-filling component in the extracellular matrix of animal tissues. Confocal fluorescence recovery after photobleaching (confocal-FRAP) was used to investigate intramolecular hydrogen bonding and electrostatic interactions in hyaluronan solutions. Self and tracer lateral diffusion coefficients within hyaluronan solutions were measured over a wide range of concentrations (c), with varying electrolyte and at neutral and alkaline pH. The free diffusion coefficient of fluoresceinamine-labeled HA of 500 kDa in PBS was 7.9 x 10(-8) cm(2) s(-1) and of 830 kDa HA was 5.6 x 10(-8) cm(2) s(-1). Reductions in self- and tracer-diffusion with c followed a stretched exponential model. Electrolyte-induced polyanion coil contraction and destiffening resulted in a 2.8-fold increase in self-diffusion between 0 and 100 mM NaCl. Disruption of hydrogen bonds by strong alkali (0.5 M NaOH) resulted in further larger increases in self- and tracer-diffusion coefficients, consistent with a more dynamic and permeable network. Concentrated hyaluronan solution properties were attributed to hydrodynamic and entanglement interactions between domains. There was no evidence of chain-chain associations. At physiological electrolyte concentration and pH, the greatest contribution to the intrinsic stiffness of hyaluronan appeared to be due to hydrogen bonds between adjacent saccharides.

Macromolecular diffusion of biological polymers measured by confocal fluorescence recovery after photobleaching.

Fluorescence recovery after photobleaching with an unmodified confocal laser scanning microscope (confocal FRAP) was used to determine the diffusion properties of network forming biological macromolecules such as aggrecan. The technique was validated using fluorescein isothiocyanate (FITC)-labeled dextrans and proteins (molecular mass 4-2000 kDa) at 25 degrees C and with fluorescent microspheres (207 nm diameter) over a temperature range of 5-50 degrees C. Lateral diffusion coefficients (D) were independent of the focus position, and the degree and extent of bleach. The free diffusion coefficient (Do) of FITC-aggrecan determined by confocal FRAP was 4.25 +/- 0.6 x 10(-8) cm2 s-1, which is compatible with dynamic laser light scattering measurements. It appeared to be independent of concentration below 2.0 mg/ml, but at higher concentrations (2-20 mg/ml) the self-diffusion coefficient followed the function D = Do(e)(-Bc). The concentration at which the self-diffusion coefficient began to fall corresponded to the concentration predicted for domain overlap. Multimolecular aggregates of aggrecan ( approximately 30 monomers) had a much lower free diffusion coefficient (Do = 6.6 +/- 1.0 x 10(-9) cm2 s-1) but showed a decrease in mobility with concentration of a form similar to that of the monomer. The method provides a technique for investigating the macromolecular organization in glycan-rich networks at concentrations close to those found physiologically.

Alpha-1-acid (AAG, orosomucoid) glycoprotein: interaction with bacterial lipopolysaccharide and protection from sepsis.

In the acute phase response to a variety of insults a rise in the levels of the acute phase proteins, including elevations of serum alpha 1 acid glycoprotein (AAG) occurs. The physiological role of AAG is unknown, however, the time course of AAG production in the acute phase response together with its strong affinity for basic compounds suggests that AAG may function as an immune modulator to bind both exogenous and endogenous inflammatory mediators. Using E. coli lipopolysaccharide (LPS), an initiator of the acute inflammatory response associated with septic shock, we demonstrate that AAG-LPS complexes can activate mouse macrophages in vitro. In a mouse animal model of sepsis, AAG was shown to protect against meningococcal endotoxin. To pursue the mechanism of AAG action we demonstrated that AAG interacts directly with LPS using dynamic light scattering particle sizing and particle mobility. We also determined the enthalpy of interaction of AAG and LPS and showed that AAG leads to agglutination of LPS impregnated rabbit red blood cells. These studies suggest that AAG may function as an immune-modulator in the acute phase response, possibly by counter-regulating the activity of macrophage pro-inflammatory cytokines.