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
Less than 1 in 10 drug candidates that enter phase 1 clinical trials actually gets approved for human use. The high failure rate is in part due to unforeseen side effects or toxicity. A better... Show moreLess than 1 in 10 drug candidates that enter phase 1 clinical trials actually gets approved for human use. The high failure rate is in part due to unforeseen side effects or toxicity. A better understanding of the role of selectivity and a better insight in the off-target activities of drug candidates could greatly aid in preventing candidates to fail for these reasons. This thesis has tried to address some aspects in this challenging part of drug discovery. The use of activity-based protein profiling as presented in Chapters 2 and 3 in drug discovery and hit-to-lead optimization, and in Chapter 5 and 6 for the interaction profiling of a drug candidate, highlights the versatility and importance of this chemical biology technique. Combined with knowledge derived from biochemical assays, such as that developed in Chapter 4, ABPP can greatly aid the medicinal chemist. The recent surge in popularity of machine learning algorithms, backed by exponential growth of the amount of biological data available, holds great promise for drug discovery. Chapters 7 and 8 showed the applicability of one such algorithm, which was able to quite reliably predict interaction profiles. The challenges in finding, determining and predicting selectivity are far from solved, but, by incrementally expanding our understanding of the binding of small molecules to their (off-)targets, truly selective inhibitors might at some point become a reality or their necessity might be mitigated. Show less
An objective of this research was to achieve direct, well-defined and non-rate-limiting electron transfer between respiratory enzymes and the electrode surface by means of 'Q-wires'. Ensuring... Show moreAn objective of this research was to achieve direct, well-defined and non-rate-limiting electron transfer between respiratory enzymes and the electrode surface by means of 'Q-wires'. Ensuring direct and fast electron transfer, these molecular wires may ultimately be part of a series of electrode surface modifications leading to the complete, stable and well-defined immobilization of an enzyme of interest. Realization of such a stable protein film may aid in the elucidation of the enzyme’s mechanism and may perhaps lead to (commercially viable) applications, such as biosensors or biofuel cells. Show less
Cells express a large array of membrane receptors on their surface that function as a communication channel between the extra- and intracellular environment of the cell. Ligands for these receptors... Show moreCells express a large array of membrane receptors on their surface that function as a communication channel between the extra- and intracellular environment of the cell. Ligands for these receptors span a wide range of biomolecules, from proteins to carbohydrates to small molecules. Some receptors are continuously recycling between the membrane and the inside of a cell, whereas others are in a steady-state at the membrane and need ligand binding for their activation and subsequent internalization. Synthetic molecules that bind to these membrane receptors can be used to either modulate their function, or to target a reporter group (i.e. a fluorescent dye) and/or a bio-active compound (drug, protein) to cells that express this receptor, ensuring delivery to a specific cell-type. The research described in this Thesis combines synthetic and biochemical methodologies to create ligands that interact selectively with membrane receptors of the GPCR and lectin-binding families. Attachment of synthetic probes, proteins or cytostatic molecules to these ligands by a variety of chemical and enzymatic methods ensured their uptake exclusively into cells that expressed the receptor of interest. Visualization of this process was enabled by the incorporation of a fluorescent dye into the final constructs. Show less
The research described in this thesis focuses on synthetic modifications of the antibiotic peptide gramicidin S (GS). The aim of the research is the development of non__toxic analogs of GS using... Show moreThe research described in this thesis focuses on synthetic modifications of the antibiotic peptide gramicidin S (GS). The aim of the research is the development of non__toxic analogs of GS using conformational and amphipathic changes induced by sugar amino acids (SAAs) and/or non__proteinogenic amino acids. The physical properties of the synthetic derivatives were studied using NMR, Molecular Modeling, Circular Dichroism, LC/MS, and X-ray techniques. Evaluation of the biological activity (antimicrobial activity and toxicity) of the peptides revealed that various synthetic derivatives appeared to be less toxic than GS. By the frequent use and misuse of antibiotics the amount of untreatable bacterial infections has increased. This research has contributed to the development of antibiotics for which resistance by bacteria should not easily occur. Show less
This thesis describes the design, synthesis and application of chemical tools for the activity-based protein profiling of proteases, with the main focus on matrix metalloproteinases (MMPs) and the... Show moreThis thesis describes the design, synthesis and application of chemical tools for the activity-based protein profiling of proteases, with the main focus on matrix metalloproteinases (MMPs) and the proteasome. The use of photoaffinity labeling is described and the thesis starts with an extensive outline of the three most often used photoreactive groups and their application in (recently published) protein profiling studies. Targeting MMPs is performed by application of photoaffinity probes, while the proteasome is commonly targeted by so-called suicide inhibitors. The last experimental chapter deals with the creation of a novel chemoselective cleavable linker and its use in the pull-down of active proteasome subunits. Show less
