Addressing selectivity of Phospholipases A2 (PLA2) towards phospholipids is crucial for defining the structural features contributing to molecular recognition between enzyme and substrate. This is fundamental to understanding enzyme-substrate binding and interactions and to develop new potent and selective PLA2 inhibitors. PLA2 enzymes are the upstream regulators of the eicosanoid pathway liberating free arachidonic acid (AA). The liberation of AA sets off a cascade of molecular events that involves downstream regulators such as cyclooxygenase (COX) and lipoxygenase (LOX), leading to inflammation (Nat. Rev. Immunol. 2015, 15, 511-523). Aspirin and other nonsteroidal anti-inflammatory drugs (NSAIDs) work by inhibiting COX and LOX which rely on PLA2 enzymes to provide them with AA. Therefore, PLA2 enzymes could potentially also be targeted to diminish inflammation at an earlier stage in the process. This study focuses on three human PLA2 enzymes: the cytosolic (cPLA2), calcium-independent (iPLA2) and lipoprotein-associated (Lp-PLA2). A lipidomics high-throughput assay was developed allowing us to define unique specificity of PLA2s towards a wide variety of phospholipids. Hydrogen/deuterium (H/D) exchange mass spectrometry was used to experimentally identify distinct PLA2 peptide regions that interact with phospholipid vesicles. Molecular dynamics simulations, guided by H/D exchange experimental data, showed that the active sites of these enzymes are allosterically regulated by membranes. Membrane phospholipids bind to allosteric sites located on the interfacial surface of PLA2s shifting their conformation from the “closed” to the “open” state (PNAS 2015, 112, E516-E525). This process facilitates extraction and binding of a phospholipid molecule into the active site. Similar methods were employed to understand the binding of various inhibitor, generate structure-activity relationships and develop new potent and selective PLA2 inhibitors. In fact, we have developed thioether fluoroketone compounds that selectively inhibit GIVA cPLA2 and GVIA iPLA2. This constitutes the first detailed study connecting molecular structure with cellular function and addressing questions related to PLA2 substrate specificity. These enzymes are implicated in chronic inflammatory diseases and understanding their interactions with substrates and inhibitors at the molecular level will allow us to identify potent and selective inhibitors to aid in the discovery of new therapeutics.

Addressing phospholipase A2 selectivity towards phospholipids: An important step for developing potent and selective inhibitors

254th National Meeting (2017)

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