5 research outputs found
Hyaluronan (HA) glycopolymers and self-assembling HA-binding peptides: a synthetic toolbox for probing HA-peptide/protein interactions and creating supramolecular HA biomaterials
PhDHyaluronan (HA) is a highly abundant anionic polysaccharide found throughout mammalian connective tissues. Unlike other glycosaminoglycans, HA is non-sulfated and its alternating chemical structure is invariable. Despite its simplicity, HA is involved in the extracellular matrix organization and many other aspects of cell behaviour. Using HA as a model polymer, we took inspiration to design and synthesize glycopolymers based on HA and self-assembling HA-binding peptides. Glycopolymers provide an alternative to conventional polysaccharides by offering the possibility to display sugars across the polymer at defined densities, while providing a robust polymer backbone rather than the glycosidic bonds, potentially forming more stable biomaterials with similar biological activity. The synthesis of alternating, homo and statistical HA neoglycopolymers was achieved via reversible addition fragmentation chain transfer (RAFT) polymerisation. The monosaccharides are grafted onto the polymers backbone by post-polymerisation click reactions to achieve the desired glycopolymers. These glycopolymers share some properties of native HA, such as water solubility and negative charge. Binding studies showed the ability of these glycopolymers to interact with HA-binding peptides and proteins, whilst not presenting any cytotoxicity behaviour. This approach provides control over the type and variation of saccharides, not be possible using natural HA, suggesting their potential use in mechanistic studies to understand HA binding processes. Peptides, on the other hand, have received increasing interest as potential biomaterials due to their inherent biocompatibility and biodegradability. Peptides can be engineered to drive their self-assembly into defined nanostructures, such as micelles and fibers. By designing HA-binding peptides with self-assembling properties, their combination with natural HA resulted in the formation of supramolecular hydrogels or membranes. Whilst the synthesis of large molecules like proteins is still a challenge, peptide synthesis is relatively simple and their self-assembly can result in nanomaterials with structures and functions resembling proteins. The synthetic molecules described in this thesis can be used as probes to better understand the role and binding of HA as well as reveal new applications.European Union (FP7), Maria Curie Career Integration Grant, SupraHApolymers (PCIG-GA2013-6318717
Proline-Rich Peptides with Improved Antimicrobial Activity against E. coli, K. Pneumoniae, and A. Baumannii.
Proline-rich antimicrobial peptides (PrAMPs) are promising agents to combat multi-drug resistant pathogens due to a high antimicrobial activity, yet low cytotoxicity. A library of derivatives of the PrAMP Bac5(1-17) was synthesized and screened to identify which residues are relevant for its activity. In this way, we discovered that two central motifs -PIRXP- cannot be modified, while residues at N- and C- termini tolerated some variations. We found five Bac5(1-17) derivatives bearing 1-5 substitutions, with an increased number of arginine and/or tryptophan residues, exhibiting improved antimicrobial activity and broader spectrum of activity while retaining low cytotoxicity toward eukaryotic cells. Transcription/translation and bacterial membrane permeabilization assays showed that these new derivatives still retained the ability to strongly inhibit bacterial protein synthesis, but also acquired permeabilizing activity to different degrees. These new Bac5(1-17) derivatives therefore show a dual mode of action which could hinder the selection of bacterial resistance against these molecules
Thyrotropin releasing hormone and its cyclised C-terminal peptide inhibit thyroglobulin release from thyroid follicular cells: evidence for acute regulation of hormone production at the thyroid gland
Supramolecular Presentation of Hyaluronan onto Model Surfaces for Studying the Behaviour of Cancer Stem Cells
The supramolecular presentation of extracellular matrix components on surfaces provides a platform for the investigation and control of cell behavior. Hyaluronan (HA) is one of the main components of the extracellular environment and has been shown to play an important role in different cancers and their progression. However, current methods of HA immobilization often require its chemical modification. Herein, a peptide-based self-assembled monolayer (SAM) is used as an anchor to immobilize unmodified HA on a bare gold surface, as demonstrated by the quartz crystal microbalance with dissipation monitoring. Peptide-HA surfaces show increased roughness and greater hydrophobicity when compared to poly-D-lysine/HA surfaces, as measured by atomic force microscopy and water contact angle, respectively. Additionally, the peptide SAM can be micro-contact printed and used to restrict the presentation of HA to specific regions, thereby creating HA patterned surfaces to examine cell behavior. When used for cell culture, these surfaces result in altered adhesion and migration of LUC4 head and neck squamous cell carcinoma cells. These biomimetic surfaces can provide insights into the role of HA in cancer and other diseases and be used as a platform for the development of cell sorting devices.C.O’M. thanks The Queen Mary Institute of Bioengineering and the
Engineering and Physical Sciences Research Council for financial support through a Ph.D. studentship (Award number 1502316).
J.B. gratefully acknowledges the financial support by Fundação para
a Ciência e a Tecnologia (FCT), I.P., through individual contract
(CEECIND/03202/2017). This work was also funded by national
funds (OE), through FCT, I.P., in the scope of the framework contract
foreseen in the numbers 4, 5, and 6 of the article 23, of the Decree-Law
57/2016, of August 29, changed by Law 57/2017, of July 19. The authors
also acknowledge the financial support from the EU-funded project
“SuprHApolymers” (PCIG14-GA-2013-631871).publishe
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The effect of lipidation and glycosylation on short cationic antimicrobial peptides
The global health threat surrounding bacterial resistance has resulted in antibiotic researchers shifting their focus away from ‘traditional’ antibiotics and concentrating on other antimicrobial agents, including antimicrobial peptides. These low molecular weight "mini-proteins" exhibit broad-spectrum activity against bacteria, including multi-drug resistant strains, viruses, fungi and protozoa and constitute a major element of the innate-immune system of many multicellular organisms. Some naturally occurring antimicrobial peptides are lipidated and/or glycosylated and almost all antimicrobial peptides in clinical use are either lipopeptides (Daptomycin and Polymyxin E and B) or glycopeptides (Vancomycin). Lipidation, glycosylation and PEGylation are an option for improving stability and activity in serum and for reducing the rapid clearing via the kidneys and liver. Two broad-spectrum antimicrobial peptides NH2-RIRIRWIIR-CONH2 (A1) and NH2-KRRVRWIIW-CONH2 (B1) were conjugated via a linker, producing A2 and B2, to individual fatty acids of C8, C10, C12 and C14 and in addition, A2 was conjugated to either glucose, N-acetyl glucosamine, galactose, mannose, lactose or polyethylene glycol (PEG). Antimicrobial activity against two Gram-positive strains (methicillin resistant Staphylococcus aureus (MRSA) and vancomycin resistant Enterococcus faecalis (VRE)) and three Gram-negative strains (Salmonella typhimurium, E. coli and Pseudomonas aeruginosa) were determined. Activity patterns for the lipidated versions are very complex, dependent on sequence, bacteria and fatty acid. Two reciprocal effects were measured; compared to the parental peptides, some combinations led to a 16-fold improvement whereas other combinations let to a 32-fold reduction in antimicrobial activity. Glycosylation decreased antimicrobial activity by 2 to 16-fold in comparison to A1, respectively on the sugar-peptide combination. PEGylation rendered the peptide inactive. Antimicrobial activity in the presence of 25% human serum of A1 and B1 was reduced 32-fold and 8-fold, respectively. The longer chain fatty acids almost completely restored this activity; however, these fatty acids increased hemolytic activity. B1 modified with C8 increased the therapeutic index by 2-fold for four bacterial strains. Our results suggest that finding the right lipid-peptide combination can lead to improved activity in the presence of serum and potentially more effective drug candidates for animal studies. Glycosylation with the optimal sugar and numbers of sugars at the right peptide position could be an alternative route or could be used in addition to lipidation to counteract solubility and toxicity issues