Inhibitor discovery of phospholipases and N-acyltransferases

Zhou, J. (2020)

Doctoral Thesis

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 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.

• PLAAT3
• PLA2G4E
• Phospholipase
• N-acyltransferase
• Inhibitor

• All authors: Zhou, J.
• Supervisor: Stelt, M. van der; Overkleeft, H.S.
• Committee: Neefjes, J.J.C.; Brouwer, J.; Aerts, J.M.F.G.; Verhelst, S.H.L.; Meijerink, J.
• Qualification: Doctor (dr.)
• Awarding Institution: Leiden Institute of Chemistry (LIC), Faculty of Science, Leiden University
• Date: 2020-11-11