Characterization of PI3Kγ enzyme to develop stilbenoid derivatives as inhibitor molecules for Alzheimer’s disease.

PI3Kγ catalyzes phosphorylation of 3-hydroxyl group of inositol ring of phosphatidylinositol and is implicated in causation of Alzheimer’s disease. Stilbenoids are naturally occurring molecules known to inhibit PI3Kγ. This necessitates biophysical characterization of PI3Kγ to develop stilbenoid-scaffold based molecules for its inhibition.

Crystal structure of human PI3Kγ was completed using MODELLER and validated using PROCHECK. Piceatannol and resveratrol, were docked to kinase domain of PI3Kγ using AutoDock Vina and complexes were subjected to molecular dynamic simulations using Desmond suite of programmes. Based on structural analysis, modified derivatives of the native molecules were designed and docked. Alongside, recombinant expression conditions for PI3Kγ were standardized using pET28a-trigger factor chaperone vector (pTf16) in BL21DE3 bacterial expression system and purified using Ni-NTA chromatography. Folded state of PI3Kγ and inhibition by stilbenoids molecules was established using ADP-Glo Kinase assay from Promega.

Kinase domain has a bi-lobar structure with ATP binding site lying in the cleft connecting the two lobes primarily composed of 12 α-helices and 8 β-strands. Piceatannol and resveratrol bind at ATP binding site, with one its rings in a position primarily occupied by adenine of ATP making a hydrogen bond with backbone of Val882. Molecules also make interactions with Lys833 and several isoleucine residues. Derivative molecules of stilbenoids also occupy ATP binding cleft and modifications result in hydrogen bonded interactions to Glu880, and ionic interactions to Lys833 and Lys808 thereby enhancing their potencies. PI3Kγ was recombinantly expressed in conditions of 24°C, 13mM L-arabinose, 0.4mM IPTG for a yield of 2mg/litre of culture. Inhibition studies showed stilbenoid molecules to be having potency in micro-nano molar range.

Array of interactions and functional inhibition assays establish stilbenoids molecules as potent inhibitors of PI3Kγ, thereby paving way for novel therapeutics in Alzheimer’s disease.