Carbohydrates, alongside proteins and nucleic acids, constitute a crucial and versatile family of biomolecules present in all life forms. They manifest as monosaccharides, oligosaccharides, and... Show moreCarbohydrates, alongside proteins and nucleic acids, constitute a crucial and versatile family of biomolecules present in all life forms. They manifest as monosaccharides, oligosaccharides, and polysaccharides, covalently bonded to proteins and fats. Carbohydrates are integral to plant and arthropod cell walls and traditionally viewed as the primary source and storage of energy, but also play a vital role in many fundamental biological processes. To study the structure and biological activity of specific carbohydrates, it is essential to synthesize them in a pure and well-defined form, as current natural extraction techniques are often limiting.The work of this thesis is focused on a specific type of glycomimetics: thiosugars, i.e. sugar analogues which have their endocylic oxygen replaced by a sulfur atom. These analogues are of interest because of their unique stability and stereoelectronic effects, which closely mimick their natural O-counterparts. The glycosidic linkage of thioglycosides (i.e. 4-S-furanosides and 5-S-pyranosides) shows improved resistance towards chemical and enzymatic hydrolysis. Show less
This thesis describes the development and optimization of the first molecular tools to study the enzyme PLA2G4E. After remaining elusive for many years, in 2016, this enzyme was discovered to be... Show moreThis thesis describes the development and optimization of the first molecular tools to study the enzyme PLA2G4E. After remaining elusive for many years, in 2016, this enzyme was discovered to be responsible for the calcium-dependent formation of N-acylphosphatidylethanolamines (NAPEs) in cells. NAPEs are low-abundant lipid species that play roles in membrane stabilization, cell signaling and homeostasis. They are well-known as precursors to the signaling lipids of the N-acylethanolamine (NAE) class, but their own biological functions remain relatively poorly understood. To find inhibitors of PLA2G4E, a focused compound library was screened in a newly developed activity-based protein profiling (ABPP) assay. Hits were identified and optimized by building structure-activity relationships (SARs) through organic synthesis and activity assays. WEN091 was identified as potent inhibitor of PLA2G4E that was able to reduce cellular NAPE levels. Cellular target engagement was confirmed by use of a tailored activity-based probe. Using these molecular tools, the relevance of NAPEs and PLA2G4E in cellular processes and disease may be elucidated. Show less
Adenosine receptors, G protein-coupled receptors (GPCRs) that are activated by the endogenous ligand adenosine, have been considered potential therapeutic targets in several disorders. To date... Show moreAdenosine receptors, G protein-coupled receptors (GPCRs) that are activated by the endogenous ligand adenosine, have been considered potential therapeutic targets in several disorders. To date however, only very few adenosine receptor modulators have made it to the market. Increased understanding of these receptors is required to improve the success rate of adenosine receptor drug discovery. To improve our understanding of receptor structure and function, over the past decades, a diverse array of molecular probes has been developed and applied. These probes, including radioactive or fluorescent moieties, have proven invaluable in GPCR research in general. Specifically for adenosine receptors, the development and application of covalent or reversible probes, whether radiolabeled or fluorescent, have been instrumental in the discovery of new chemical entities, the characterization and interrogation of adenosine receptor subtypes, and the study of adenosine receptor behavior in physiological and pathophysiological conditions. This review summarizes these applications, and also serves as an invitation to walk another mile to further improve probe characteristics and develop additional tags that allow the investigation of adenosine receptors and other GPCRs in even finer detail. Show less
Drug development is a time- and resource-consuming process that starts with the discovery and validation of a (protein) target that contributes to pathogenesis or disease progression. One of the... Show moreDrug development is a time- and resource-consuming process that starts with the discovery and validation of a (protein) target that contributes to pathogenesis or disease progression. One of the essential steps in this process is to validate that pharmacological modulation (e.g. inhibition) of the target leads to the desired phenotype, a process which is collectively referred to as target validation. Target validation heavily relies on the availability of suitable chemical tools to study engagement of the compound to the intended biological target. The development of selective chemical tools can be challenging to achieve due to the off-target activity towards structurally and/or functionally related homologs, e.g. other members within the same protein class. The field of chemical genetics combines the specificity of genetics with benefits of acute, pharmacological modulation by small molecules. This thesis describes chemical genetic approaches that can be used for target engagement and target validation studies of two different enzyme classes: kinases and serine hydrolases. Show less
In this thesis, the discovery and optimization is described of chemical tools to study the N-acylethanolamine (NAE) biosynthetic pathway. In particular, two enzymes – N-acylphosphatidylethanolamine... Show moreIn this thesis, the discovery and optimization is described of chemical tools to study the N-acylethanolamine (NAE) biosynthetic pathway. In particular, two enzymes – N-acylphosphatidylethanolamine phospholipase D (NAPE-PLD) and phospholipase A and acyltransferase 2 (PLAAT2) – were targeted, which produce NAEs or their NAPE precursors, respectively. So far, genetic KO models have not been able to fully elucidate the complexity of NAE biosynthesis, possibly due to long-term compensatory effects. By blocking these enzymes in an acute fashion, the contributory role of NAPE-PLD and PLAAT2 with regard to NAE formation can be assessed across specific cells and tissues. To identify inhibitors for these enzymes, high throughput screening or focused-library screening approaches were applied. Using structure-activity relationship studies, initial hits were optimized to potent inhibitors, possessing cellular and/or in vivo efficacy. On-target confirmation was achieved by employing photoaffinity labeling or activity-based protein profiling. Cellular and/or in vivo activity of the described inhibitors was confirmed with targeted lipidomics experiments. To conclude, the herein developed NAPE-PLD and PLAAT2 inhibitors (LEI-401 and LEI-301, respectively) are suitable starting points to investigate the biological consequences of depleting the NAE tone, which may be useful in pathological conditions such as obesity, metabolic syndrome, chronic liver disease and cancer. Show less
The cannabinoid receptor type 2 (CB2R) is associated with several inflammatory diseases with an unmet medical need (e.g. Alzheimers, multiple sclerosis, reumatoid arthritis). Development of... Show moreThe cannabinoid receptor type 2 (CB2R) is associated with several inflammatory diseases with an unmet medical need (e.g. Alzheimers, multiple sclerosis, reumatoid arthritis). Development of new chemical biology strategies to study this protein is essential to aid future development of drugs for these diseases. Show less
In this thesis the behavior and functionality of peptide amphiphiles at the surface of bilayer vesicles is examined. By controlling the behavior of the surface bound peptides, I was able to... Show moreIn this thesis the behavior and functionality of peptide amphiphiles at the surface of bilayer vesicles is examined. By controlling the behavior of the surface bound peptides, I was able to construct assemblies which could: 1) release their content (triggered by pH), 2) fuse in a targeted and controlled manner or 3) dock to cells and zebrafish embryos Show less
This Thesis aims at the development of novel subunit selective inhibitors of the proteasome. -Three vinyl sulfone analogues of three epoxyketone containing inhibitors described in literature are... Show moreThis Thesis aims at the development of novel subunit selective inhibitors of the proteasome. -Three vinyl sulfone analogues of three epoxyketone containing inhibitors described in literature are synthesised and characterised. The nature of the electrophile determines subunit selectivity of the inhibitor. -Ten Michael acceptors are coupled to three peptoid tails yielding thirty peptide-like compounds. None of the peptoid Michael acceptors are efficient proteasome inhibitors. Using the Staudinger-Bertozzi ligation followed by affinity purification, tryptic digestion of the isolated proteins and LC/MS/MS identification, the targets of two compounds in HEK293T and RAW264.7 cells are determined. -Syringolins are potent proteasome inhibitors found in nature. A syringolin structural motif was built in peptoid epoxyketones and vinyl sulfones yielding a 16-membered library of proteasome inhibitors. The distance between the urea and electrophile dictates subunit selectivity. -Three cyclooctynes are compared to the Staudinger-Bertozzi two-step labelling strategy. Cyclooctynes quantitatively convert azide labelled proteasomes at a lower concentration than the Staudinger-Bertozzi phosphane but cyclooctyne two-step labelling is associated with tremendous background labelling. -A library of peptoid vinyl sulfones with basic amino acid side chains is synthesised. A compound with two benzylamine residues is a potent and _2 selective proteasome inhibitor. Attachment of a BODIPY fluorophore yielded a _2 selective probe. Show less
