Computer-aided drug design guided by hydrogen/deuterium exchange mass spectrometry: A powerful combination for the development of potent and selective inhibitors of Group VIA calcium-independent phospholipase A₂

Potent and selective inhibitors for phospholipases A₂ (PLA₂) are useful for studying their intracellular functions. PLA₂ enzymes liberate arachidonic acid from phospholipids activating eicosanoid pathways that involve cyclooxygenase (COX) and lipoxygenase (LOX) leading to inflammation. Anti-inflammatory drugs target COX and LOX; thus, PLA₂ can also be targeted to diminish inflammation at an earlier stage in the process. This paper describes the employment of enzymatic assays, hydrogen/deuterium exchange mass spectrometry (DXMS) and computational chemistry to develop PLA₂ inhibitors. Beta-thioether trifluoromethylketones (TFKs) were screened against human GVIA calcium-independent, GIVA cytosolic and GV secreted PLA₂s. These compounds exhibited inhibition toward Group VIA calcium-independent PLA₂ (GVIA iPLA₂), with the most potent and selective inhibitor 3 (OTFP) obtaining an X_I(50) of 0.0002 mole fraction (IC₅₀ of 110 nM). DXMS binding experiments in the presence of OTFP revealed the peptide regions of GVIA iPLA₂ that interact with the inhibitor. Molecular docking and dynamics simulations in the presence of a membrane were guided by the DXMS data in order to identify the binding mode of OTFP. Clustering analysis showed the binding mode of OTFP that occupied 70% of the binding modes occurring during the simulation. The resulted 3D complex was used for docking studies and a structure–activity relationship (SAR) was established. This paper describes a novel multidisciplinary approach in which a 3D complex of GVIA iPLA₂ with an inhibitor is reported and validated by experimental data. The SAR showed that the sulfur atom is vital for the potency of beta-thioether analogues, while the hydrophobic chain is important for selectivity. This work constitutes the foundation for further design, synthesis and inhibition studies in order to develop new beta-thioether analogues that are potent and selective for GVIA iPLA₂ exclusively.