Understanding the stereospecific interactions of 3-deoxyphosphatidylinositol derivatives with the PTEN phosphatase domain

Abstract

PTEN is an important control element of PI3K/AKT signaling involved in controlling the processes of embryonic development, cell migration and apoptosis. While its dysfunction is implicated in a large fraction of cancers, PTEN activity in the same pathway may also contribute to metabolic syndromes such as diabetes. In those cases, selective inhibitors of PTEN may be useful. A new class of chiral PTEN inhibitors based on the 3-deoxy-phosphatidylinositol derivatives was recently identified [Wang et al. (2008) J. Am. Chem. Soc. 130, 7746]. However, lack of detailed understanding of protein-ligand interactions has hampered efforts to develop effective agonists or antagonists of PTEN. Here, we use computational modeling to characterize the interactions of the diverse 3-deoxyphosphatidylinositol inhibitors with the PTEN protein. We show that, while each of the compounds binds with the inositol headgroup inserting into the proposed active site of the PTEN phosphatase domain, hydrogen bonding restrictions lead to distinct binding geometries for ligand pairs of opposite chirality. We furthermore demonstrate that the binding modes differ primarily in the orientation of acyl tails of the ligands and that the activity of the compounds is primarily controlled by the effectiveness of tail-protein contacts. These findings are confirmed by binding affinity calculations which are in good agreement with experiment. Finally, we show that while more potent D-series ligands bind in a manner similar to that of the native substrate, an alternate hydrophobic pocket suitable for binding the opposite chirality L-series inhibitors exists, offering the possibility of designing highly selective PTEN- targeting compounds.

Phospholipid-binding Sites of Phosphatase and Tensin Homolog (PTEN)

“…followed a previously published protocol with minor modifications (13,14) that are summarized below. The plasmid was transformed into Escherichia coli BL21-CodonPlus (DE3)-RIL cells from Stratagene, and the cells were grown at 37°C until the A 600 reached 0.8.…”

“…All mutations were confirmed by DNA sequencing (GeneWiz). Protein expression and purification of the mutant PTEN proteins followed the procedure for the wild type protein (13). The typical yield was 1-1.5 mg/liter culture.…”

“…Triton X-100 is a nonionic detergent that we have previously shown to be able to reduce the inhibition potency of shortchain deoxy-PI derivatives by binding to a hydrophobic site adjacent to the active site (AHS) and/or forming micelles that alter the on/off rates of the PI derivative in the active site (13). The presence of the nonionic detergent in the diC 8 PI(3)P/ diC 8 PI(4,5)P 2 assay system had little effect on the PIP 2 activation of PTEN (Fig.…”

“…Tartrate occupied the active site and was replaced with inositol 1,3,4,5-tetrakisphosphate in silico to have a sense of substrate binding. Molecular dynamics simulations of different dioctanoyl phosphatidylinositols bound to the catalytic domain of PTEN suggested that adjacent to the active site is another region that can play a role in monomer ligand binding (13). Those simulations suggested that there are specific interactions between the acyl chain of these lipids and the ␤2-␣1 loop containing Arg-47.…”

“…Those simulations suggested that there are specific interactions between the acyl chain of these lipids and the ␤2-␣1 loop containing Arg-47. Mutants of Arg-47 showed much lower activity compared with the wild type protein regardless of the identity of the mutated amino acid, indicating the importance of this particular residue for substrate binding and/or catalysis (13). The interactions of the ␤2-␣1 loop could facilitate the binding of several 3-deoxy-PI derivatives that inhibit enzyme activity (14).…”