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
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
