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The development of functionalised stapled peptides as chemical tools to modulate biological processes in platelets and as novel antimicrobial therapeutics targeting Pseudomonas aeruginosa
Peptides are useful modulators of protein-protein interactions (PPIs) and cellular membranes, both of which are traditionally challenging to target using small molecules. Often therapeutic peptides suffer from issues including poor proteolytic stability. Two-component peptide stapling can improve stability and enable facile peptide functionalisation. This thesis describes the development of functionalised stapled peptides for two biological applications.
1. The development of stapled peptides as chemical tools to investigate PPIs in human platelets
Platelets are a vital, anuclear component of blood. The Bcl-2 family of proteins are known to be key mediators of apoptosis in nucleated cells, however the role of each Bcl-2 protein in platelet apoptosis and activation is unknown. Recently, stapled peptides were deemed useful chemical tools for studying PPIs in platelets. In this section, three polyarginine-functionalised stapled peptides were developed as Bcl-2 PPI inhibitors. These novel chemical tools are anticipated to provide valuable insight into the roles of Bcl-2 PPIs in platelet modulation, which could ultimately lead to new therapeutic targets.
2. The development of cleavable stapled peptide-drug conjugates to target Pseudomonas aeruginosa
Antimicrobial resistance (AMR) is a major healthcare threat, and Gram-negative bacteria such as Pseudomonas aeruginosa pose a significant therapeutic challenge. Antimicrobial peptides disrupt bacterial membranes, however functionalised two-component stapled antimicrobial peptides (STAMPs) are underexplored. This section describes the development of a STAMP-drug conjugate, constructed by functionalisation of the STAMP staple with a small molecule antibiotic, attached via a β-lactamase-cleavable motif. The resulting conjugate combines two mechanisms of action, which is a validated strategy for overcoming AMR. To this end, two novel, unfunctionalised STAMPs were identified that exhibited good minimum inhibitory concentrations against P. aeruginosa (64 μg/mL) and selectivity over a Gram-positive strain, from a panel of seven novel STAMPs. Subsequently, a tractable synthesis of the proposed functionalised STAMP was investigated. It is hypothesised that the cleavable STAMP-drug conjugate will enable infection-controlled drug-release and dual-targeting of P. aeruginosa
Microscopy and chemical analyses reveal flavone-based woolly fibres extrude from micron-sized holes in glandular trichomes of Dionysia tapetodes.
BackgroundDionysia tapetodes, a small cushion-forming mountainous evergreen in the Primulaceae, possesses a vast surface-covering of long silky fibres forming the characteristic "woolly" farina. This contrasts with some related Primula which instead form a fine powder. Farina is formed by specialized cellular factories, a type of glandular trichome, but the precise composition of the fibres and how it exits the cell is poorly understood. Here, using a combination of cell biology (electron and light microscopy) and analytical chemical techniques, we present the principal chemical components of the wool and its mechanism of exit from the glandular trichome.ResultsWe show the woolly farina consists of micron-diameter fibres formed from a mixture of flavone and substituted flavone derivatives. This contrasts with the powdery farina, consisting almost entirely of flavone. The woolly farina in D. tapetodes is extruded through specific sites at the surface of the trichome's glandular head cell, characterised by a small complete gap in the plasma membrane, cell wall and cuticle and forming a tight seal between the fibre and hole. The data is consistent with formation and thread elongation occurring from within the cell.ConclusionsOur results suggest the composition of the D. tapetodes farina dictates its formation as wool rather than powder, consistent with a model of thread integrity relying on intermolecular H-bonding. Glandular trichomes produce multiple wool fibres by concentrating and maintaining their extrusion at specific sites at the cell cortex of the head cell. As the wool is extensive across the plant, there may be associated selection pressures attributed to living at high altitudes
Microscopy and chemical analyses reveal flavone-based woolly fibres extrude from micron-sized holes in glandular trichomes of Dionysia tapetodes
Abstract Background Dionysia tapetodes, a small cushion-forming mountainous evergreen in the Primulaceae, possesses a vast surface-covering of long silky fibres forming the characteristic “woolly” farina. This contrasts with some related Primula which instead form a fine powder. Farina is formed by specialized cellular factories, a type of glandular trichome, but the precise composition of the fibres and how it exits the cell is poorly understood. Here, using a combination of cell biology (electron and light microscopy) and analytical chemical techniques, we present the principal chemical components of the wool and its mechanism of exit from the glandular trichome. Results We show the woolly farina consists of micron-diameter fibres formed from a mixture of flavone and substituted flavone derivatives. This contrasts with the powdery farina, consisting almost entirely of flavone. The woolly farina in D. tapetodes is extruded through specific sites at the surface of the trichome’s glandular head cell, characterised by a small complete gap in the plasma membrane, cell wall and cuticle and forming a tight seal between the fibre and hole. The data is consistent with formation and thread elongation occurring from within the cell. Conclusions Our results suggest the composition of the D. tapetodes farina dictates its formation as wool rather than powder, consistent with a model of thread integrity relying on intermolecular H-bonding. Glandular trichomes produce multiple wool fibres by concentrating and maintaining their extrusion at specific sites at the cell cortex of the head cell. As the wool is extensive across the plant, there may be associated selection pressures attributed to living at high altitudes
Stapled peptides as a new technology to investigate protein-protein interactions in human platelets.
Platelets are blood cells with numerous crucial pathophysiological roles in hemostasis, cardiovascular thrombotic events and cancer metastasis. Platelet activation requires the engagement of intracellular signalling pathways that involve protein-protein interactions (PPIs). A better understanding of these pathways is therefore crucial for the development of selective anti-platelet drugs. New strategies for studying PPIs in human platelets are required to overcome limitations associated with conventional platelet research methods. For example, small molecule inhibitors can lack selectivity and are often difficult to design and synthesise. Additionally, development of transgenic animal models is costly and time-consuming and conventional recombinant techniques are ineffective due to the lack of a nucleus in platelets. Herein, we describe the generation of a library of novel, functionalised stapled peptides and their first application in the investigation of platelet PPIs. Moreover, the use of platelet-permeable stapled Bim BH3 peptides confirms the part of Bim in phosphatidyl-serine (PS) exposure and reveals a role for the Bim protein in platelet activatory processes. Our work demonstrates that functionalised stapled peptides are a complementary alternative to conventional platelet research methods, and could make a significant contribution to the understanding of platelet signalling pathways and hence to the development of anti-platelet drugs.British Heart Foundation project grant to Dr. Nicholas Pugh (PG/14/47/30912)
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MRC
Royal Society