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
In this thesis an activity-based probe was discovered that could visualize the activity of PLAATs. With an optimized gel-based ABPP assay in hand, screening of a compound library led to the... Show moreIn this thesis an activity-based probe was discovered that could visualize the activity of PLAATs. With an optimized gel-based ABPP assay in hand, screening of a compound library led to the discovery of alpha-ketoamides as a hit for PLAAT3. Through extensive structural modifications of the hit, LEI110 was identified as the most potent inhibitor (Ki = 20nM) for PLAAT3. LEI110 reduced cellular arachidonic acid levels in PLAAT3 overexpressing U2OS cells and oleic acid-induced steatosis in human HepG2 cells. Gel-based ABPP and chemical proteomics showed that LEI110 is a selective pan-inhibitor of the Hrasls-family of thiol hydrolases (i.e. PLAAT2, PLAAT3 and PLAAT5). LEI110 could be an excellent starting point for the structure-based drug development of novel molecular therapies for obesity and/or common cold. In addition, a competitive, gel-based ABPP method for PLA2G4E using TAMRA-FP was successfully developed and applied to screen a focused library of lipase inhibitors. This resulted in the discovery of two clusters of inhibitors with different scaffolds. Optimization of the potency and selectivity of the inhibitors is required to the study of the biological role of PLA2G4E in an acute and dynamic setting with these novel tools. Together these novel chemical tools and methods will allow for a better understanding of the biosynthesis of the NAPEs and to study their biological role. Show less
