Illuminating N-acylethanolamine biosynthesis with new chemical tools

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 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.Molecular Physiolog