Phospholipase A2, group XVI (PLA2G16) is a thiol hydrolase from the HRASLS family that regulates lipolysis in adipose tissue and has been identified as a host factor enabling the cellular entry of picornaviruses. Chemical tools are essential to visualize and control PLA2G16 activity, but they have not been reported to date. Here, we show that MB064, which is a fluorescent lipase probe, also labels recombinant and endogenously expressed PLA2G16. Competitive activity-based protein profiling (ABPP) using MB064 enabled the discovery of alpha-ketoamides as the first selective PLA2G16 inhibitors. LEI110 was identified as a potent PLA2G16 inhibitor ( K(i) = 20 nM) that reduces cellular arachidonic acid levels and oleic acid-induced lipolysis 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., PLA2G16, HRASLS2, RARRES3 and iNAT). Molecular dynamic simulations of LEI110 in the reported crystal structure of PLA2G16 provided insight in the potential ligand-protein interactions to explain its binding mode. In conclusion, we have developed the first selective inhibitor that can be used to study the cellular role of PLA2G16.
Endocannabinoids, an important class of signaling lipids involved in health and disease, are predominantly synthesized and metabolized by enzymes of the serine hydrolase superfamily. Activity-based protein profiling (ABPP) using fluorescent probes, such as fluorophosphonate (FP)-TAMRA and beta-lactone-based MB064, enables drug discovery activities for serine hydrolases. FP-TAMRA and MB064 have distinct, albeit partially overlapping, target profiles but cannot be used in conjunction due to overlapping excitation/emission spectra. We therefore synthesized a novel FP-probe with a green BODIPY as a fluorescent tag and studied its labeling profile in mouse proteomes. Surprisingly, we found that the reporter tag plays an important role in the binding potency and selectivity of the probe. A multiplexed ABPP assay was developed in which a probe cocktail of FP-BODIPY and MB064 visualized most endocannabinoid serine hydrolases in mouse brain proteomes in a single experiment. The multiplexed ABPP assay was employed to profile endocannabinoid hydrolase inhibitor activity and selectivity in the mouse brain.
