The Leptospermum scoparium (Manuka)-Specific Nectar and Honey Compound 3,6,7-Trimethyllumazine (LepteridineTM) That Inhibits Matrix Metalloproteinase 9 (MMP-9) Activity.

3,6,7-trimethyllumazine (Lepteridine™) is a newly discovered natural pteridine derivative unique to Manuka (Leptospermum scoparium) nectar and honey, with no previously reported biological activity. Pteridine derivative-based medicines, such as methotrexate, are used to treat auto-immune and inflammatory diseases, and Manuka honey reportedly possesses anti-inflammatory properties and is used topically as a wound dressing. MMP-9 is a potential candidate protein target as it is upregulated in recalcitrant wounds and intestinal inflammation. Using gelatin zymography, 40 µg/mL LepteridineTM inhibited the gelatinase activities of both pro- (22%, p < 0.0001) and activated (59%, p < 0.01) MMP-9 forms. By comparison, LepteridineTM exerted modest (~10%) inhibition against a chromogenic peptide substrate and no effect against a fluorogenic peptide substrate. These findings suggest that LepteridineTM may not interact within the catalytic domain of MMP-9 and exerts a negligible effect on the active site hydrolysis of small soluble peptide substrates. Instead, the findings implicate fibronectin II domain interactions by LepteridineTM which impair gelatinase activity, possibly through perturbed tethering of MMP-9 to the gelatin matrix. Molecular modelling analyses were equivocal over interactions at the S1' pocket versus the fibronectin II domain, while molecular dynamic calculations indicated rapid exchange kinetics. No significant degradation of synthetic or natural LepteridineTM in Manuka honey occurred during simulated gastrointestinal digestion. MMP-9 regulates skin and gastrointestinal inflammatory responses and extracellular matrix remodelling. These results potentially implicate LepteridineTM bioactivity in Manuka honey's reported beneficial effects on wound healing via topical application and anti-inflammatory actions in gastrointestinal disorder models via oral consumption.

Amyloid Fibrils from Hemoglobin.

Amyloid fibrils are a class of insoluble protein nanofibers that are formed via the self-assembly of a wide range of peptides and proteins. They are increasingly exploited for a broad range of applications in bionanotechnology, such as biosensing and drug delivery, as nanowires, hydrogels, and thin films. Amyloid fibrils have been prepared from many proteins, but there has been no definitive characterization of amyloid fibrils from hemoglobin to date. Here, nanofiber formation was carried out under denaturing conditions using solutions of apo-hemoglobin extracted from bovine waste blood. A characteristic amyloid fibril morphology was confirmed by transmission electron microscopy (TEM) and atomic force microscopy (AFM), with mean fibril dimensions of approximately 5 nm diameter and up to several microns in length. The thioflavin T assay confirmed the presence of ß-sheet structures in apo-hemoglobin fibrils, and X-ray fiber diffraction showed the characteristic amyloid cross-ß quaternary structure. Apo-hemoglobin nanofibers demonstrated high stability over a range of temperatures (-20 to 80 °C) and pHs (2-10), and were stable in the presence of organic solvents and trypsin, confirming their potential as nanomaterials with versatile applications. This study conclusively demonstrates the formation of amyloid fibrils from hemoglobin for the first time, and also introduces a cost-effective method for amyloid fibril manufacture using meat industry by-products.

Proteins as supramolecular building blocks: Nterm-Lsr2 as a new protein tecton.

Proteins hold great promise in forming complex nanoscale structures which could be used in the development of new nanomaterials, devices, biosensors, electronics, and pharmaceuticals. The potential to produce nanomaterials from proteins is well supported by the numerous examples of self-assembling proteins found in nature. We have explored self-assembling proteins for use as supramolecular building blocks, or tectons, specifically the N-terminal domain of Lsr2, Nterm-Lsr2. A key feature of this protein is that it undergoes self-assembly via proteolytic cleavage, thereby allowing us to generate supramolecular assemblies in response to a specific trigger. Herein, we report the effects of pH and protein concentration on the oligomerization of Nterm-Lsr2. Furthermore, via protein engineering, we have introduced a new trigger for oligomerization via enteropeptidase cleavage. The new construct of Nterm-Lsr2 can be activated and assembled in a controlled fashion and provides some ability to alter the ratio of higher ordered structures formed.

Ovine forestomach matrix biomaterial is a broad spectrum inhibitor of matrix metalloproteinases and neutrophil elastase.

Proteases play a critical role in the ordered remodelling of extracellular matrix (ECM) components during wound healing and tissue regeneration. However, the usually ordered proteolysis is compromised in chronic wounds due to over-expression and high concentrations of matrix metalloproteinase's (MMPs) and neutrophil elastase (NE). Ovine forestomach matrix (OFM) is a decellularised extracellular matrix-based biomaterial developed for tissue regeneration applications, including the treatment of chronic wounds, and is a heterogeneous mixture of ECM proteins and proteoglycans that retains the native structural and functional characteristics of tissue ECM. Given the diverse molecular species present in OFM, we hypothesised that OFM may contain components or fragments that inhibit MMP and NE activity. An extract of OFM was shown to be a potent inhibitor of a range of tissue MMPs (IC50 s = 23 ± 5 to 115 ± 14 µg/ml) and NE (IC50 = 157 ± 37 µg/ml), and was more potent than extracts prepared from a known protease modulating wound dressing. The broad spectrum activity of OFM against different classes of MMPs (i.e. collagenases, gelatinases and stromelysins) may provide a clinical advantage by more effectively addressing the protease imbalance seen in chronic wounds.

Biochemical and structural characterisation of dehydroquinate synthase from the New Zealand kiwifruit Actinidia chinensis.

One of the novel aspects of kiwifruit is the presence of a high level of quinic acid which contributes to the flavour of the fruit. Quinic acid metabolism intersects with the shikimate pathway, which is responsible for the de novo biosynthesis of primary and secondary aromatic metabolites. The gene encoding the enzyme which catalyses the second step of the shikimate pathway, dehydroquinate synthase (DHQS), from the New Zealand kiwifruit Actinidia chinensis was identified, cloned and expressed. A. chinensis DHQS was activated by divalent metal ions, and was found to require NAD(+) for catalysis. The protein was crystallised and the structure was solved, revealing a homodimeric protein. Each monomer has a NAD(+) binding site nestled between the distinct N- and C-terminal domains. In contrast to other microbial DHQSs, which show an open conformation in the absence of active site ligands, A. chinensis DHQS adopts a closed conformation. This is the first report of the structure of a DHQS from a plant source.

Quantification of in vitro and in vivo angiogenesis stimulated by ovine forestomach matrix biomaterial.

Ovine forestomach matrix (OFM) biomaterial acts as a biomimetic of native extracellular matrix (ECM) by providing structural and functional cues to orchestrate cell activity during tissue regeneration. The ordered collagen matrix of the biomaterial is supplemented with secondary ECM-associated macromolecules that function in cell adhesion, migration and communication. As angiogenesis and vasculogenesis are critical processes during tissue regeneration we sought to quantify the angiogenic properties of the OFM biomaterial. In vitro studies demonstrated that soluble OFM components stimulated human umbilical vein endothelial cell (HUVEC) migration and increased vascular sprouting from an aorta. Blood vessel density and branch points increased in response to OFM in an ex ovo chicken chorioallantoic membrane (CAM) assay. The OFM biomaterial was shown to undergo remodeling in a porcine full-thickness excisional model and gave rise to significantly more blood vessels than wounds treated with small intestinal submucosa decellularized ECM or untreated wounds.

Expression, Purification, and Characterisation of Dehydroquinate Synthase from Pyrococcus furiosus.

Dehydroquinate synthase (DHQS) catalyses the second step of the shikimate pathway to aromatic compounds. DHQS from the archaeal hyperthermophile Pyrococcus furiosus was insoluble when expressed in Escherichia coli but was partially solubilised when KCl was included in the cell lysis buffer. A purification procedure was developed, involving lysis by sonication at 30°C followed by a heat treatment at 70°C and anion exchange chromatography. Purified recombinant P. furiosus DHQS is a dimer with a subunit Mr of 37,397 (determined by electrospray ionisation mass spectrometry) and is active over broad pH and temperature ranges. The kinetic parameters are K(M) (3-deoxy-D-arabino-heptulosonate 7-phosphate) 3.7 µM and k(cat) 3.0 sec(-1) at 60°C and pH 6.8. EDTA inactivates the enzyme, and enzyme activity is restored by several divalent metal ions including (in order of decreasing effectiveness) Cd(2+), Co(2+), Zn(2+), and Mn(2+). High activity of a DHQS in the presence of Cd(2+) has not been reported for enzymes from other sources, and may be related to the bioavailability of Cd(2+) for P. furiosus. This study is the first biochemical characterisation of a DHQS from a thermophilic source. Furthermore, the characterisation of this hyperthermophilic enzyme was carried out at elevated temperatures using an enzyme-coupled assay.

Fluorinated substrates result in variable leakage of a reaction intermediate during catalysis by dehydroquinate synthase.

Incubation of (3S)-3-fluoro-3-deoxy-D-arabino-heptulosonate 7-phosphate with dehydroquinate (DHQ) synthase from three phylogenetically distinct sources resulted in the production of (6S)-6-fluoroDHQ and its epimer 1-epi-(6S)-6-fluoroDHQ. The differences in the product ratios of the reactions catalysed by each enzyme imply that 1-epi-(6S)-6-fluoroDHQ formation occurs by an unusual partial leakage of a reaction intermediate from the enzyme.

Biophysical characterization of ovine forestomach extracellular matrix biomaterials.

Ovine forestomach matrix (OFM) is a native and functional decellularized extracellular matrix biomaterial that supports cell adhesion and proliferation and is remodeled during the course of tissue regeneration. Small angle X-ray scattering demonstrated that OFM retains a native collagen architecture (d spacing = 63.5 ± 0.2 nm, orientation index = 20°). The biophysical properties of OFM were further defined using ball-burst, uniaxial and suture retention testing, as well as a quantification of aqueous permeability. OFM biomaterial was relatively strong (yield stress = 10.15 ± 1.81 MPa) and elastic (modulus = 0.044 ± 0.009 GPa). Lamination was used to generate new OFM-based biomaterials with a range of biophysical properties. The resultant multi-ply OFM biomaterials had suitable biophysical characteristics for clinical applications where the grafted biomaterial is under load.

A functional extracellular matrix biomaterial derived from ovine forestomach.

Extracellular matrix (ECM) based biomaterials have an established place as medical devices for wound healing and tissue regeneration. In the search for biomaterials we have identified ovine forestomach matrix (OFM), a thick, large format ECM which is biochemically diverse and biologically functional. OFM was purified using an osmotic process that was shown to reduce the cellularity of the ECM and aid tissue delamination. OFM produced using this technique was shown to retain residual basement membrane components, as evidence by the presence of laminin and collagen IV. The collagenous microarchitecture of OFM retained many components of native ECM including fibronectin, glycosaminoglycans, elastin and fibroblast growth factor basic. OFM was non-toxic to mammalian cells and supported fibroblast and keratinocyte migration, differentiation and infiltration. OFM is a culturally acceptable alternative to current collagen-based biomaterials and has immediate clinical applications in wound healing and tissue regeneration.

Synthesis and SAR investigations for novel melanin-concentrating hormone 1 receptor (MCH1) antagonists part 2: A hybrid strategy combining key fragments of HTS hits.

A novel series of melanin-concentrating hormone (MCH1) receptor antagonists based on combining key fragments from the high-throughput screening (HTS) hits compound 2 (SNAP 7941) and compound 5 (chlorohaloperidol) are described. The resultant analogs, exemplified by compounds 11a-11h, 15a-15h, and 16a-16g, were evaluated in in vitro and in vivo assays for their potential in treatment of mood disorders. From further SAR investigations, N-(3-{1-[4-(3,4-difluorophenoxy)benzyl]-4-piperidinyl}-4-methylphenyl)-2-methylpropanamide (16g, SNAP 94847) was identified to be a high affinity and selective ligand for the MCH1 receptor. Compound 16g also shows good oral bioavailability (59%) and exhibits a brain/plasma ratio of 2.3 in rats. Compound 16g showed in vivo inhibition of a centrally induced MCH-induced drinking effect and exhibited a dose-dependent anxiolytic effect in the rat social interaction model.