Specificity mechanism of Group VIA calcium-independent phospholipase A2 toward truncated-oxidized phospholipids and its application for specific inhibitor design

Abstract

Phospholipase A₂ (PLA₂) constitutes a superfamily of enzymes that hydrolyze the sn-2 fatty acyl chain of glycerophospholipids. Polyunsaturated fatty acids (PUFAs) are preferentially attached at the sn-2 position of glycerophospholipids and are easily truncated by oxidation. The truncated-oxidized phospholipids (tr-oxPLs) trigger various cellular responses, and PLA₂s may play a critical role in the metabolism of the tr-oxPLs by removing the oxidized sn-2 chain. In the present study, we demonstrated using an in vitro lipidomics assay that Group VIA calcium-independent PLA₂ (GVIA iPLA₂) showed high activity toward phosphatidylcholine with a 9-oxononanoyl chain, but not with an azelaoyl chain on the sn-2 position. We conducted molecular dynamics simulations which revealed that the hydrophilicity of the sn-2 acyl chain critically affects the binding of the substrate in the active site. Based on the unique specificity of GVIA iPLA₂ toward tr-oxPLs, we synthesized an oxidatively modified inhibitor (GK766) for GVIA iPLA₂, aiming for improvement of its selectivity and/or potency. As we expected, the modified inhibitor improved its selectivity of GVIA iPLA₂ compared to the unmodified inhibitor (GK187), although the inhibitory effect became somewhat weaker. More importantly, we demonstrated that GK766 induces cell death by ferroptosis more effectively than GK187 using an erythroleukemia cell line. In the present study, we have further defined the unique substrate specificity of GVIA iPLA₂ toward tr-oxPLs and its molecular mechanism. Furthermore, we have developed a novel specificity-based inhibitor that induces ferroptosis demonstrating that using substrate selectivity helps in developing more effective therapeutics.