Fluoroketone Inhibition of Ca²⁺-Independent Phospholipase A₂ through Binding Pocket Association Defined by Hydrogen/Deuterium Exchange and Molecular Dynamics

Abstract

The mechanism of inhibition of group VIA Ca2+-independent phospholipase A2 (iPLA2) by fluoroketone (FK) ligands is examined by a combination of deuterium exchange mass spectrometry (DXMS) and molecular dynamics (MD). Models for iPLA2 were built by homology with the known structure of patatin and equilibrated by extensive MD simulations. Empty pockets were identified during the simulations and studied for their ability to accommodate FK inhibitors. Ligand docking techniques showed that the potent inhibitor 1,1,1,3-tetrafluoro-7-phenylheptan-2-one (PHFK) forms favorable interactions inside an active-site pocket, where it blocks the entrance of phospholipid substrates. The polar fluoroketone headgroup is stabilized by hydrogen bonds with residues Gly486, Gly487, and Ser519. The nonpolar aliphatic chain and aromatic group are stabilized by hydrophobic contacts with Met544, Val548, Phe549, Leu560, and Ala640. The binding mode is supported by DXMS experiments showing an important decrease of deuteration in the contact regions in the presence of the inhibitor. The discovery of the precise binding mode of FK ligands to the iPLA2 should greatly improve our ability to design new inhibitors with higher potency and selectivity.

Homologous ligands accommodated by discrete conformations of a buried cavity

“…For signal transduction, the importance of such conformational changes has long been recognized (8), and has been emphasized by recent experimental (9) and computational studies (10). Accordingly, molecular dynamics simulations of protein-ligand complexes are now widely considered for ligand design (11)(12)(13)(14)(15)(16).…”

Calcium-independent phospholipases A2 and their roles in biological processes and diseases

“…Recently developed and awaiting characterization is an even more selective inhibitor (GK187) of iPLA 2 ␤ ( 162 ). On-going deuterium exchange mass spectrometry and molecular dynamics analyses suggest that FKGK inhibitor binding to iPLA 2 ␤ causes changes in the loops surrounding the active site of iPLA 2 ␤ in the catalytic domain, blocking access to phospholipid substrates and reducing solvent accessibility ( 163 ). As the development of chemical inhibitors continues, newer structurally dissimilar and smaller compounds ( 164 ) with even greater selectivity for iPLA 2 ␤ are forthcoming, as described at the 6th International Conference on PLA 2 s in 2015 (Kokotos et al, unpublished observations).…”

A novel dimeric active site and regulation mechanism revealed by the crystal structure of iPLA2β

“…Alterations in iPLA 2  function have demonstrated its role in multiple human pathologies including cardiovascular disease 1,[25][26][27] , cancer [28][29][30][31] , diabetes 32,33 , muscular dystrophy 34 , nonalcoholic steatohepatitis 35 and antiviral responses 36 . Correspondingly, novel inhibitors of iPLA 2  have been sought for therapeutic applications 37,38 . Highly selective mechanism-based fluoroketone inhibitors were designed 37,39,40 and successfully applied in mouse models of diabetes 41 and multiple sclerosis 42 .…”

“…Correspondingly, novel inhibitors of iPLA 2  have been sought for therapeutic applications 37,38 . Highly selective mechanism-based fluoroketone inhibitors were designed 37,39,40 and successfully applied in mouse models of diabetes 41 and multiple sclerosis 42 . Recently, numerous mutations have been discovered in patients with neurodegenerative disorders such as infantile neuroaxonal dystrophy (INAD) [43][44][45] and PD [3][4][5][6][7][8][9][10][11] .…”

“…The first crystal structure of iPLA 2 β has revealed several unexpected features underlying its enzymatic activity, mechanisms of regulation and structural domains potentially involved in tissue-specific localization. Previous computer modeling studies used the patatin structure and proposed an interfacial activation mechanism whereby interaction with membrane leads to opening of a closed active site 37 . In the iPLA 2  crystal structure, the active peer-reviewed) is the author/funder.…”