Activity-based protein profiling identifies α-ketoamides as inhibitors for Phospholipase A2 Group XVI

Zhou, J.; Mock, E.D.; Martella, A.; Kantae, V.; Di, X.; Burggraaff, L.; Baggelaar, M.P.; Al-Ayed, K.; Bakker, A.; Florea, B.I.; Grimm, S.H.; Dulk, H. den; Li, C.T.; Mulder, L.; Overkleeft, H.S.; Westen, G.J.P. van; Hankemeier, T.; Stelt, M. van der

doi:doi:10.1021/acschembio.8b00969

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 have not been reported to date. Here we show that MB064, a fluorescent lipase probe, also labels recombinant and endogenously expressed PLA2G16. Competitive activity-based protein profiling (ABPP) using MB064 enabled the discovery of α-ketoamides as the first selective PLA2G16 inhibitors. LEI110 was identified as a potent PLA2G16 inhibitor (Ki = 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...Show morePhospholipase 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 have not been reported to date. Here we show that MB064, a fluorescent lipase probe, also labels recombinant and endogenously expressed PLA2G16. Competitive activity-based protein profiling (ABPP) using MB064 enabled the discovery of α-ketoamides as the first selective PLA2G16 inhibitors. LEI110 was identified as a potent PLA2G16 inhibitor (Ki = 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.Show less

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Activity-based protein profiling identifies α-ketoamides as inhibitors for Phospholipase A2 Group XVI