The endocannabinoid receptors CB1R and CB2R are involved in a plethora of processes, and consequently are involved in many pathological conditions. Their wide distribution makes the CBRs both an... Show moreThe endocannabinoid receptors CB1R and CB2R are involved in a plethora of processes, and consequently are involved in many pathological conditions. Their wide distribution makes the CBRs both an interesting therapeutic target and hard to study. Additional chemical tools are required to study and understand the function and mechanism of CB1R and CB2R. This thesis describes the development of several such tools to improve our insight in the (pathological) roles of the receptors in order to develop novel and improved therapeutics. First evaluation of three dimensional ligand-CB2R complexes made and analysed with Cryo-EM are described. Hotspots that potentially generate selectivity between CB1R and CB2R are evaluated with point-mutations in vitro. Consequently describes the development of the first tools, two-step bifunctional probes based on LEI-121 and LEI 102, is described. As two-step probes are not compatible with every assay, the toolbox is expanded with a one-step fluorescent probe. Briefly touching upon CB1R, ligands were designed with negatively charged phosphonium groups that are potentially selective for mtCB1R. Show less
This thesis introduces the concept of "physics-based inverse design", working on the notion that the physical driving forces governing functionality are inherently encoded in independently... Show moreThis thesis introduces the concept of "physics-based inverse design", working on the notion that the physical driving forces governing functionality are inherently encoded in independently parameterized energy functions, which can be resolved through the use of inverse design strategies.The thesis describes the development of EVO-MD, a Python-based implementation of the physics-based inverse design concept. EVO-MD is capable of automatically setting-up, performing, and analyzing molecular dynamics simulations, allowing for the evolutionary optimization of complex and dynamic features in peptides. Examples of such applications include the optimization of lipid composition and curvature sensors, and the development of peptides with antiviral properties. Show less
The research presented in this thesis explores the chemotherapeutic potential of metal-based compounds as chemotherapy agents, with an initial focus on the synthesis and DNA interaction studies of... Show moreThe research presented in this thesis explores the chemotherapeutic potential of metal-based compounds as chemotherapy agents, with an initial focus on the synthesis and DNA interaction studies of platinum and palladium compounds utilizing the [Pt(bapbpy)]2+ scaffold. The study identifies intercalation as the primary mechanism of action for these complexes. Furthermore, it provides a detailed structure-activity relationship analysis, highlighting the critical role of the complex's protonation state in influencing its biological activity and efficacy. Subsequently, the study delves into photoactivated chemotherapy (PACT) using ruthenium (II) complexes, where light activation of ruthenium complexes enables targeted drug delivery to tumor cells, thereby reducing adverse effects. This research emphasizes the development of ruthenium-based compounds that can photorelease a DNA repair inhibitor, specifically targeting the RAD51 protein, essential for Homologous Recombination (HR). By disrupting the DNA repair mechanisms in cancer cells, this approach seeks to enhance the cytotoxicity of the therapy and address drug resistance. Show less
β-Lactamases are enzymes that can break down β-lactam substrates, such as antibiotics, preventing the use of these antibiotics for the treatment of various infectious diseases. However, some... Show moreβ-Lactamases are enzymes that can break down β-lactam substrates, such as antibiotics, preventing the use of these antibiotics for the treatment of various infectious diseases. However, some compounds, β-lactamase inhibitors, can block these enzymes allowing for possible treatments using a combination of antibiotic and inhibitor. BlaC is the β-lactamase of Mycobacterium tuberculosis, the bacteria that cause tuberculosis, and is used as a model for protein evolution. To understand if and how BlaC can develop resistance against certain inhibitors we studied the evolutionary adaptability of this enzyme. We used laboratory evolution and various biochemical techniques to characterize several mutations in BlaC and subsequently tested the effect of combining mutations. One of the findings is that BlaC can easily become less sensitive to the inhibitor sulbactam by partially blocking the entrance to the active site. Interestingly, this was accompanied by increased sensitivity to another inhibitor, avibactam, that could not be compensated for by other mutations.Generally, Escherichia coli bacteria are used to test the effects of BlaC variants in cells, as they are easy and safe to use in the lab. We show that results obtained for E. coli can be extrapolated to conditions that resemble tuberculosis disease in humans: the M. marinum infection model of zebrafish. All these findings are of interest for the future development of combination therapies to treat tuberculosis. Show less
Several single-stranded RNA viruses make use of Xrn1-resistant RNAs in their 3’ untranslated regions of their genome RNAs in order to increase their pathogenicity. This thesis focuses on two types... Show moreSeveral single-stranded RNA viruses make use of Xrn1-resistant RNAs in their 3’ untranslated regions of their genome RNAs in order to increase their pathogenicity. This thesis focuses on two types of Xrn1-resistant RNAs: those involving the “coremin” motif (xrRNAC) and those found in members of the Flaviviridae family (xrRNAF). While the structure for xrRNAFs has been solved, the xrRNAC structure is yet elusive. Therefore, we employed systematic mutational analysis in order to identify the features that are involved in halting the 5’-3’ exoribonuclease Xrn1 by xrRNAC. This led to the identification of novel variations of xrRNAC in viral families that were not yet known to employ an xrRNA. Regarding xrRNAF, we investigated their distribution and variability throughout the Flaviviridae family, and concluded that a universal xrRNAF structure is responsible for stalling Xrn1. Furthermore, the work in this thesis expands on the known, potential functions of xrRNAs by showing how xrRNAC is able to both inhibit scanning ribosomes and promote frameshifting. Show less
This thesis describes the synthesis and biological evaluation of TLR2/6, TLR4, TLR7/8 and TLR9 ligands, of which the activity can be conditionally controlled. Trans-cyclooctenes are used to shield... Show moreThis thesis describes the synthesis and biological evaluation of TLR2/6, TLR4, TLR7/8 and TLR9 ligands, of which the activity can be conditionally controlled. Trans-cyclooctenes are used to shield the ligand's moiety critical for TLR activation, which can be removed fast and quantitatively by the addition of tetrazine derivatives. Photocages are used in parallel for the same purpose, which can be removed by exposure to low-energetic UV-light. A new photocage has been developed that can be decorated on two positions, i.e. it is bifunctional. This photocage has been used to synthesize a proteasome inhibitor-doxorubicin conjugate, in which the photocage bisects the two groups, which induces apoptosis in targeted multiple myeloma cells with an acquired resistance for proteasome inhibitors. The photocage was also used to synthesize a probe that enables targeting and isolation of CD8+ T cells in viable human melanoma and non-squamous cell lung cancer tissues. Show less
Global healthcare is on the verge of an antibiotic availability crisis as bacteria have evolved resistance to nearly all known antibacterials. Identifying new antibiotics that operate via novel... Show moreGlobal healthcare is on the verge of an antibiotic availability crisis as bacteria have evolved resistance to nearly all known antibacterials. Identifying new antibiotics that operate via novel modes-of-action is therefore of high priority.This thesis contains two drug discovery projects, originating from a antibacterial screen of a compound library. In both projects chemical hits are first structurally optimized, after which their mode-of-action is determined.The first project entails optimizing a hit with potency against MRSA into a submicromolar active antibiotic. By using a chemical proteomics approach, the targets of this compound were elucidated, along with the targets that are most important in its antibacterial activity.The second project concerns Gram-negative bacteria, where a hit molecule is optimized into the conformationally restricted LEI-800. The target of LEI-800 is found to be DNA gyrase, a common antibiotic target. However, it is that LEI-800 inhibits DNA gyrase differently, and more potently, than the status quo. Show less
Supramolecular polymers are class of materials that are formed by non-covalent interactions such as hydrogen bonding, π-π interactions, electrostatic interactions and the hydrophobic effect. The... Show moreSupramolecular polymers are class of materials that are formed by non-covalent interactions such as hydrogen bonding, π-π interactions, electrostatic interactions and the hydrophobic effect. The design and development of supramolecular polymers in aqueous solution gained a particular attention for the wide variety of applications in the biomedical field. In water, the self-assembly of well-defined nanostructures is mainly determined by the combination of hydrophobic effect with hydrogen bonding interactions in the monomer design. When squaramide-based monomer self-assemble, the formation of stable nanostructures in water is determined by the formation of directional hydrogen bonds which are strengthened by the partial aromatic character of the squaramide. In this thesis, the self-assembly properties of a panel of squaramide-based monomers is examined in aqueous solution through modulating the monomer chemical structure, co-assembly and introduction of light responsive chemistries. Show less
Ice, the solid state of water, plays an important role on our planet as well as the entire universe.Despite the fact that an individual water molecule has a very simple structure, its chemical... Show moreIce, the solid state of water, plays an important role on our planet as well as the entire universe.Despite the fact that an individual water molecule has a very simple structure, its chemical bonding in the solid phase can be surprisingly complex.Nowadays, atomistic computational models allow to describing and understanding these properties in a way that has not been possible for a long time.Chemical interactional potentials are at the heart of these atomistic models.In increasing order of complexity, these potentials range from simple pair potentials over polarizable force fields up to density functional theory (DFT).It is an ongoing scientific challenge is to improve and test these potentials.This thesis attempts to provide some answers to the following research questions:(i) How important is the contribution of zero-point energy to thermodynamical properties of ice phases?(ii) How accurately do available interaction potentials allow to model (small) differences between H2O and D2O ices related to nuclear quantum effects?(iii) Do interaction potentials need to be improved when nuclear quantum effects in ice are taken into account? Show less
Het proefschrift omschrijft de chemische synthese van fenolische glycolipiden van verschillende mycobacteriën met het doel om deze te kunnen gebruiken voor immunologisch onderzoek.
Glycoside hydrolases (glycosidases/GHs) are widely abundant enzymes in all kingdoms of life and are important biocatalysts that catalyze the hydrolysis of glycosidic linkages in oligo... Show moreGlycoside hydrolases (glycosidases/GHs) are widely abundant enzymes in all kingdoms of life and are important biocatalysts that catalyze the hydrolysis of glycosidic linkages in oligo/polysaccharides, glycoproteins and glycolipids with tremendous efficiency. Abnormal glycosidase activity is intimately associated with a variety of human diseases. Overexpression of heparanase, for example, is implicated in almost all cancers examined, and correlates with increased tumor size, tumor angiogenesis, enhanced metastasis and poor prognosis. Specific inhibitors of glycosidases are of great value, not only because they can serve as useful biological tools to study the catalytic machinery, mechanism and itinerary of target enzymes by crystal structure analysis of (covalent) inhibitor-enzyme complexes, but also because they may act as starting points for the development of therapeutic drugs for the treatment of glycosidase-mediated diseases. Additionally, covalent mechanism-based inhibitors have been used as scaffolds for the development of activity-based probes (ABPs) which allow profiling of glycosidases in complex biological systems. The research described in this dissertation focus on the development and biochemical evaluation of covalent inhibitors and ABPs for retaining endo- and exo-glycosidases including starch-degrading enzymes and human lysosomal β-glucocerebrosidase (GBA), as well as the synthesis of a panel of uronic acid-type 1-N-iminosugars as potential competitive heparanase inhibitors. Show less
Artificial photosynthesis has recognised potential to produce green and sustainable fuels from earth-abundant resources such as water, carbon dioxide (CO2), and sunlight. In an artificial... Show moreArtificial photosynthesis has recognised potential to produce green and sustainable fuels from earth-abundant resources such as water, carbon dioxide (CO2), and sunlight. In an artificial photosynthetic system, two half-reactions, such as water oxidation and proton reduction or CO2 reduction, have to be combined. To achieve such a system, it is crucial to have: a) efficient light-harvesting by the photosensitiser, b) stable catalysts for the oxidation and the reduction reaction, c) unidirectional proton and electron transport between the oxidation and the reduction site, ideally by a recyclable electron relay, d) efficient charge separation, and e) a strong, photostable membrane that does not leak molecular components. In natural photosynthesis, these requirements are achieved altogether using compartmentalisation, which consists in embedding the key components of the system, i.e. for green plants the oxygen evolving complex, photosystem I and II, and the natural electron relays, around the lipid bilayer of the thylakoid membrane. The use of spherical lipid membranes (such as liposomes) as biological mimics of the thylakoid membrane is a promising approach to confine half-reactions, facilitate charge separation, and avoid charge recombination and other undesired side-reactions. In the research described in this thesis, it was attempted to realise a full artificial photosynthetic system based on liposomes and several of the key intermediate steps were achieved: 1) unidirectional electron transfer across a liposomal membrane from an electron donor encapsulated in the interior of the liposome to an electron acceptor located outside (Chapter 2), and 2) photocatalytic reduction of CO2 (Chapter 3) and of protons (Chapter 4) at the surface of liposomes. Special attention was paid in Chapter 2 and Chapter 5 to the question of the (photo)stability of the membrane and light-induced leakage. Show less
This thesis presents the first synthetic peptides ADP-ribosylated on serine, threonine, tyrosine, arginine and cysteine. Besides synthetic peptides, this thesis discusses the first synthetic route... Show moreThis thesis presents the first synthetic peptides ADP-ribosylated on serine, threonine, tyrosine, arginine and cysteine. Besides synthetic peptides, this thesis discusses the first synthetic route towards ADP-ribosylated nucleic acids. Furthermore, two photoaffinity probes for PARP1 have been developed and assessed in living cells and two activity based probes have been synthesized, designed for CD38. Show less
The redox-conversion reaction of metal-disulfide and metal-thiolate complexes are important, as they may shed light on electron-transfer reactions that often occur in Nature. Despite their... Show moreThe redox-conversion reaction of metal-disulfide and metal-thiolate complexes are important, as they may shed light on electron-transfer reactions that often occur in Nature. Despite their importance, very few examples have been reported. In addition to that, there is a limited understanding of how the coordination environment of the metal ion affects this reaction. In this thesis, our investigation was set based on the ligand-field theory to determine its correlation with the redox-conversion reactions in cobalt-based systems. Our experiments revealed that using an exogenous ligand with a strong ligand-field character may induce the redox conversion from cobalt(II)-disulfide complexes to cobalt(III)-thiolate complexes. Using this knowledge, the possibility of the redox-conversion reaction was also extended from cobalt(II)-diselenide to cobalt(III)-selenolate complexes.Finally, we have come up with several conclusions about the redox-conversion reactions of the cobalt(II)-dichalcogenide complexes. It was revealed that the conversion is affected by the ligand-field strength of the dichalcogenide ligand. The smaller ligand-field strength can be counterbalanced with the introduction of the strong auxiliary ligand. Lastly, the cleanliness of the conversion depends on the magnitude of the overall ligand-field splitting energy of the complex. Show less
Artificial photosynthesis (AP) is one of the scientific challenges that could help us achieving a global “carbon neutral” society. Photocatalytic water splitting is considered as the first... Show moreArtificial photosynthesis (AP) is one of the scientific challenges that could help us achieving a global “carbon neutral” society. Photocatalytic water splitting is considered as the first challenge of AP, which contains two half reactions: water oxidation and hydrogen evolution. It is widely accepted that a photocatalytic system needs a minimum of three components: a photosensitizer (PS), a catalyst (Cat) and a sacrificial electron donor or acceptor (SE). In such a photocatalytic system, at least three electron-transfer steps can be identified: one between the SE and the excited PS (PS*), one between the photo-reduced or photo-oxidized PS and the Cat, and one between the Cat and its substrate. This thesis on the one hand focused on developing improved molecular components for the two half reactions of water splitting in purely homogeneous systems. On the other hand optimized photocatalytic systems with balances between the driving force of electron transfer from the SE to the PS*, and that of electron transfer between the catalyst and the oxidized or reduced photosensitizer (PS+ or PS–). Show less
The research described in this Thesis was aimed at designing and synthesizing nature-inspired compounds as part of TB vaccine discovery. A variety of synthetic analogues of mycobacterial cell wall... Show moreThe research described in this Thesis was aimed at designing and synthesizing nature-inspired compounds as part of TB vaccine discovery. A variety of synthetic analogues of mycobacterial cell wall components, from peptide and glycolipid antigens to glycolipid PAMPs has been accessed. Evaluation of the immune stimulatory activity of the novel compounds in combination with preliminary immunization studies in vivo, suggested the potential of selected synthetic conjugates as single molecule vaccines against TB. Further research is needed to verify the efficacy of these vaccine modalities. Show less
Evolution acts via mutations in amino acid sequences. Substitution of essential amino acids leads to a nonfunctional protein. Thus, the number of essential residues is limited by evolutionary... Show moreEvolution acts via mutations in amino acid sequences. Substitution of essential amino acids leads to a nonfunctional protein. Thus, the number of essential residues is limited by evolutionary pressure. The roles of all non-catalytic essential residues in class A β-lactamases are described with a large-scale experiments, as well as specific functions of a few residues. The results show that residues close to the active site and farther away have different reasons for being essential. Show less
This thesis focuses on the synthesis, characterization and performance towards CO2 electroreduction of mono and bi-metallic particles based on p-block metals. With an industrial perspective in mind... Show moreThis thesis focuses on the synthesis, characterization and performance towards CO2 electroreduction of mono and bi-metallic particles based on p-block metals. With an industrial perspective in mind, we try to synthesize particulate, high surface area materials with clean, scalable synthesis methods where possible and test their performance in H-Cell and gas diffusion electrode flow cell configurations. With a combination of characterization techniques, we find possible explanations for the catalytic behaviors. Show less
This thesis has shed light on some of the ways in which the local electrolyte composition can differ from the bulk and how these changes in the local reaction environment can determine the activity... Show moreThis thesis has shed light on some of the ways in which the local electrolyte composition can differ from the bulk and how these changes in the local reaction environment can determine the activity and/or selectivity of two important electrocatalytic reactions, namely, electrochemical CO2 reduction reaction (CO2RR) and hydrogen evolution reaction (HER). Show less
