Given the accelerating appearance of antimicrobial resistance, there is an urgent need for more fundamental research into novel antibiotic strategies. The work in this thesis helps to address this... Show moreGiven the accelerating appearance of antimicrobial resistance, there is an urgent need for more fundamental research into novel antibiotic strategies. The work in this thesis helps to address this global problem by developing new antibiotic compounds, inspired by the antibacterial mechanisms of the natural antibiotic bacitracin. By unravelling the unique mechanism of action that bacitracin employs, we discovered that the inclusion of a small hydrophobic group in key locations of the molecule results in a dramatic enhancement of antibacterial activity, in some cases more than 100 times more potent than bacitracin. Crucially we found that the most potent analogues are particularly active against antibiotic-resistant bacteria including those bearing clinically challenging resistance genes. In doing so we have developed potent next-generation variants of this classic antibiotic and have taken important steps in the fight against antimicrobial resistance. Show less
Viral diseases constitute one of the major challenges in modern medicine. Membrane-targeting have broad-spectrum potential and could distinguish viral membranes from the host cell plasma membrane... Show moreViral diseases constitute one of the major challenges in modern medicine. Membrane-targeting have broad-spectrum potential and could distinguish viral membranes from the host cell plasma membrane by the difference in membrane curvature. In this thesis, we used a combination of molecular dynamics simulations and an evolutionary algorithm to inverse design such curvature-sensing antiviral peptides, purely from physical principles. Show less
A structural investigation of coiled coil peptides used as membrane fusogens, mimicing naturally occuring coiled-coil fusion proteins. Synthetic modifications have been made to alter lipid... Show moreA structural investigation of coiled coil peptides used as membrane fusogens, mimicing naturally occuring coiled-coil fusion proteins. Synthetic modifications have been made to alter lipid attachment, secondary structure and to insert photoactive azobenzene moieties for active control over coiled coil structure. Finally, the underlying photocontrol mechanism investigated in coiled coil peptides is extended to beta-structured peptides, and was shown to be universally applicable. Show less
Large parts of the research described in this Thesis aims at the development of oligopeptide-masked toxins and their in situ immunoproteasome-mediated activation. Chapter 2 focuses on the... Show moreLarge parts of the research described in this Thesis aims at the development of oligopeptide-masked toxins and their in situ immunoproteasome-mediated activation. Chapter 2 focuses on the development of selective fluorogenic substrates for each of the iCP and cCP proteasome subunits. Chapter 3 describes the utilization of proteasome cleavable linkers in order to release a toxic entity in immunoproteasome expressing cells. By selecting oligopeptide sequences of previously described immunoproteasome-selective fluorogenic substrates and inhibitors, and linking these to doxorubicin via a cleavable linker, new constructs were obtained. Chapter 4 describes an immunoproteasome inhibitor-doxorubicin conjugate that targets multiple myeloma derived cells and releases doxorubicin upon low-dose photon irradiation. Chapter 5 describes how a trans-cyclooctene (TCO) is utilized for the release of a toxic payload via a click to release reaction in conjunction with selective proteasome targeting. TCOs were used in order to facilitate toxin activation via an inverse electron demanding Diels-Alder reaction with tetrazine as a dienophile. Chapter 6 gives insight in the development of pan-immuno subunit selective proteasome inhibitors based on P3 and P4 scaffold exchanges. Finally, the research described in this thesis is summarized in Chapter 7, and future prospects based the presented results are discussed. Show less
Membrane-exposed sulfatides are proposed to contribute to P-selectin-dependent platelet aggregation. Here, we demonstrated that P-selectin-mediated platelet aggregation on a collagen-coated surface... Show moreMembrane-exposed sulfatides are proposed to contribute to P-selectin-dependent platelet aggregation. Here, we demonstrated that P-selectin-mediated platelet aggregation on a collagen-coated surface under flow indeed depended on sulfatides and that this interaction differed considerably from the interaction of P-selectin with P-selectin Glycoprotein Ligand-1 (PSGL-1), which underlies leukocyte-endothelium adhesion.\n= 7-12 μM).\nOur data suggest that the sulfatide/P-selectin interaction implicates multiple binding pockets, which only partly overlap with that of PSGL-1. These observations open ways to selectively interfere with sulfatide/P-selectin-dependent platelet aggregation without affecting PSGL-1-dependent cell adhesion. Show less
Membrane fusion is a vital process in living organisms and is mediated by zipper-like proteins. This thesis describes the use of coiled-coil peptides, derived from these zipper-like proteins, to... Show moreMembrane fusion is a vital process in living organisms and is mediated by zipper-like proteins. This thesis describes the use of coiled-coil peptides, derived from these zipper-like proteins, to mediate fusion of liposomes. Designed mutations in the peptides and in the peptide-liposome connection influences the rate and the efficiency of the fusion process. The efficiency of the fusion process is found to be very dependent on peptide structures and peptide-membrane interactions. Crucial for efficient fusion are subtle membrane interactions and very helical peptide structures. This improves our understanding of the fusion mechanism and enables the rational design of new and better fusogens. The used peptides are also able to mediate fusion between liposomes and cells and are currently used to develop drug delivery systems. Show less
Proteasomes are multi-protein, multi-catalytic complexes responsible for the degradation of 80-90% of the proteins inside eukaryotic cells. Proteasomes contain a cylindrical 20S core particle (CP)... Show moreProteasomes are multi-protein, multi-catalytic complexes responsible for the degradation of 80-90% of the proteins inside eukaryotic cells. Proteasomes contain a cylindrical 20S core particle (CP) and one or two 19S regulatory particles (RP). The constitutive proteasome core particle (cCP), which is expressed in all mammalian tissues, contains three catalytically active subunits, namely β1c, β2c and β5c. Lymphoid cells express another proteasome core particle known as the immunoproteasome (iCP). In iCPs, β1c, β2c and β5c are replaced by β1i, β2i and β5i. The research described in this thesis reports on the development of new subunit-selective inhibitors and activity-based probes, on the development of an assay to simultaneously monitor all cCP and iCP catalytic activities and on the development of a method that reports on CP catalytic active subunit composition. The tools that stem from the work described in this thesis can now be used to unravel the role of each individual catalytic subunit in a chemical genetics setting (selective and (near) complete inhibition of each subunit), and to clarify the role of mCPs, in, for instance, antigen presentation and cancer. Furthermore, these tools could possibly serve as leads in the discovery of agents for future treatment of cancer and autoimmune diseases. Show less
Xin, B.T.; Bruin, G. de; Plomp, J.W.; Florea, B.I.; Marel, G.A. van der; Overkleeft, H.S. 2016
Seasonal influenza epidemics lead to severe flu in 3 to 5 million individuals and emerging pandemic influenza strains pose an even greater threat to society. We describe a clinical trial in which... Show moreSeasonal influenza epidemics lead to severe flu in 3 to 5 million individuals and emerging pandemic influenza strains pose an even greater threat to society. We describe a clinical trial in which vaccine-specific responses were measured during two consecutive influenza seasons, including the season in which the 2009 pandemic virus emerged, and showed that these vaccines induced both humoral and cellular responses. However, these responses are unlikely to be protective against newly emerging strains, due to the variable nature of influenza virus. Therefore, the other chapters in this thesis describe concepts within peptide-based vaccination strategies, which is one method to induce responses to highly conserved sequences of influenza virus. We evaluated a concept based on long peptides directed to highly conserved B and T cell epitopes and showed that this vaccine was capable of inducing both humoral and cellular immune responses. Furthermore, the vaccine provided partial protection against infection in an animal model. We also described another successful strategy to enhance the immunogenicity of minimal peptides by modification of these peptides and proceeded with formulations to improve delivery of these minimal peptides. Altogether, the findings in this thesis may contribute to the development of the next generation influenza vaccines. Show less
Nature__s own building block, peptide/protein derived materials have been of great interest for supramolecular chemists. The amino acids in peptides/proteins are linked via amide bonds, which makes... Show moreNature__s own building block, peptide/protein derived materials have been of great interest for supramolecular chemists. The amino acids in peptides/proteins are linked via amide bonds, which makes them more stable against degradation as compared to other natural materials such as oligonucleotides. Peptides adopt a secondary structure which is determined by their amino acid sequence resulting in a structure with a specific fold like a beta sheet, a helix or a random coil conformation.These secondary structures can govern the supra-molecular structure of the macromolecule to achieve specific function. Peptides can be short, such as dipeptides or as long as a small protein, which are able to selfassemble into a designed nanostructure and thus providing a wide choice of biomaterials for a chemical biologist. In last decade, peptides have been shown to have great versatility and inherent high affinity for their target to carry out various functions which is the scope of this thesis presented here. Show less
Het transplanteren van stamcellen van een gezonde donor, allogene stamceltransplantatie, is een potentieel genezende behandeling van hematologische maligniteiten en aangeboren hematopo_etische... Show moreHet transplanteren van stamcellen van een gezonde donor, allogene stamceltransplantatie, is een potentieel genezende behandeling van hematologische maligniteiten en aangeboren hematopo_etische ziekten. In de periode na allogene stamceltransplantatie kunnen ernstige virale infecties optreden. Omdat het herstel van virus-specifieke T cellen gepaard gaat met bescherming tegen virale ziekte na stamceltransplantatie, is het overbrengen van virus-specifieke donor T cellen naar de ontvanger een aantrekkelijke strategie ter voorkoming of ter behandeling van virale ziekten. In dit proefschrift zijn methoden onderzocht om virus-specifieke CD8+ en CD4+ T cellen in perifeer bloed te activeren om detectie en isolatie van deze specifieke cellen mogelijk te maken. De resultaten beschreven in dit proefschrift onderbouwen de rationale voor het toepassen van cellulaire immuuntherapie voor virale infecties na stamceltransplantatie. Daarnaast zijn effici_nte en klinisch toepasbare methoden ontwikkeld om gecombineerde CD8+ en CD4+ T cel lijnen te produceren met hoge specificiteit voor meerdere peptiden van verschillende virale eiwitten. Deze methoden zijn breed toepasbaar en maken het mogelijk om de klinische waarde van cellulaire immuuntherapie voor behandeling van virale infecties na allogene stamceltransplantatie in klinische studies vast te stellen. Show less
This Thesis deals with the design, synthesis and biological evaluation of peptide-based drugs. In chapters 1-4, the development of peptides derived from natural gluten which can serve as drugs to... Show moreThis Thesis deals with the design, synthesis and biological evaluation of peptide-based drugs. In chapters 1-4, the development of peptides derived from natural gluten which can serve as drugs to combat the symptoms of celiac disease is described. These symptoms are caused by a misdirected immune response towards dietary gluten in genetically predisposed individuals. Peptides which can inhibit this immune response possibly can be used as an addition to the gluten-free diet, which is the only therapy available today. In Chapters 5 and 6, the synthesis and biological evaluation analogs of the antibiotic peptide Gramicidin S is described. Gramicidin S is a naturally occurring antibiotic peptide. It is very effective against bacteria, but also exhibits toxicity towards human red blood cells which limits its use to topical applications. Analogs of this natural peptide may lead to efficient antibiotics which are broadly applicable. Show less
Of the various biomolecular building blocks in use in nature, coiled-coil forming peptides are amongst those with the most potential as building blocks for the synthetic self-assembly of... Show moreOf the various biomolecular building blocks in use in nature, coiled-coil forming peptides are amongst those with the most potential as building blocks for the synthetic self-assembly of nanostructures. Native coiled coils have the ability to function in, and influence, complex systems composed of multiple building blocks. However, there have only been a limited number of synthetic coiled-coil assemblies that mimic native coiled coils by incorporating multiple assembling components. This thesis represents efforts at extending this aspect of coiled-coil self-assembly. In order to achieve this, a range of hybrid molecules were synthesized which combine coiled-coil peptides with a hydrophobic component. In this way the highly specific coiled-coil self-assembly is juxtaposed with the non-specific, but structure-inducing aggregation of the hydrophobic section. This thesis asked simple questions: can coiled coils function when covalently attached to large hydrophobic blocks? How large can the hydrophobic blocks be? Can coiled coils function when incorporated noncovalently with a supramolecular assembly? By answering these fundamental questions the possibilities of prescriptive self-assembly have been probed and expanded, novel preparative methods have been developed, and specific applications have arisen. Show less
The first half of this thesis describes the synthesis of several conformationally restricted alkylated and bicyclic sugar amino acids (SAAs). The second half of the thesis describes the application... Show moreThe first half of this thesis describes the synthesis of several conformationally restricted alkylated and bicyclic sugar amino acids (SAAs). The second half of the thesis describes the application of the SAAs and their intermediates presented in the first half, as components of tools applied for the probing of biological systems. Show less
Intracellular proteins are degraded by the proteasome. The resulting protein fragments can be regarded as waste, but it is clear that peptides play an important role in several processes, like the... Show moreIntracellular proteins are degraded by the proteasome. The resulting protein fragments can be regarded as waste, but it is clear that peptides play an important role in several processes, like the immune response. Peptides are destroyed very rapidly and irrespectively of their sequence. A peptide's length appeared to be important as the half-life increases when additional amino acids are present at the amino-terminus. As exception dibasic N-terminal peptides appeared to be more stable than other peptides, resulting in overrepresentation by HLA-B27 molecules that bind preferably binds dibasic N-terminal peptides. Cross-presentation of peptides from infected cells by professional antigen presenting cells (APC) is crucial for a proper immune response because they express the required co-stimulatory molecules. The exact mode of antigen transfer is largely unknown, and we describe a novel pathway for cross-presentation, by passive diffusion of peptides through gap junctions from infected cells to APCs Apoptosis-derived antigens have been shown to be a major source of cross-presented antigens. Peptides from apoptotic cells can be presented by the cell itself, or transferred to healthy neighboring cells and be presented by MHC class I molecules of these cells, thereby facilitating cross-presentation, through gap junction mediated intercellular peptide transfer. Show less
