The PIK3CA gene is one of the most frequently mutated oncogenes
in human cancers. It encodes p110a the catalytic subunit of
phosphatidylinositol 3-kinase alpha (PI3Ka which activates signaling
cascades leading to cell proliferation, survival, and cell growth. The
most frequent mutation in PIK3CA is H1047R, which results in enzymatic
overactivation. Understanding how the H1047R mutation causes the enhanced
activity of the protein in atomic detail is central to developing
mutant-specific therapeutics for cancer. To this end, Surface Plasmon
Resonance (SPR) experiments and Molecular Dynamics (MD) simulations were
carried out for both wild-type (WT) and H1047R mutant p110a proteins. An
expanded positive charge distribution on the membrane binding regions of
the mutant with respect to the WT protein is observed through MD
simulations, which justifies the increased ability of the mutated protein
variant to bind to membranes rich in anionic lipids in our SPR
experiments. Our results further support an auto-inhibitory role of the
C-terminal tail in the WT protein, which is abolished in the mutant
protein due to loss of crucial intermolecular interactions. Moreover,
Functional Mode Analysis reveals that the H1047R mutation alters the
twisting motion of the N-lobe of the kinase domain with respect to the
C-lobe and shifts the position of the conserved P-loop residues in the
vicinity of the active site. These findings demonstrate the dynamical and
structural differences of the two proteins in atomic detail and propose a
mechanism of overactivation for the mutant protein. The results may be
further utilized for the design of mutant-specific PI3Ka inhibitors that
exploit the altered mutant conformation.
http://journals.plos.org/ploscompbiol/article?id=10.1371/journal.pcbi.1003895
Allosteric modulators offer a novel approach for kinase inhibition
because they target less conserved binding sites compared to the active
site thus, higher selectivity may be obtained. PIK-108, a known pan
phosphoinositide 3-kinase (PI3K) inhibitor, was recently detected to
occupy a non-ATP binding site in the PI3Ka C-lobe. This newly identified
pocket is located close to residue 1047, which is frequently mutated in
human cancers (H1047R). In order to assess the interactions, stability,
and any possible allosteric effects of this inhibitor on PI3Ka, extensive
molecular dynamics (MD) simulations in aqueous solution were performed
for the wild type (WT) human, WT murine, and H1047R human mutant PI3Ka proteins with PIK-108 placed in both catalytic and non-ATP sites. We
verify the existence of the second binding site in the vicinity of the
hotspot H1047R PI3Ka mutation through binding site identification and MD
simulations. PIK-108 remains stable in both sites in all three variants
throughout the course of the simulations. We demonstrate that the pose
and interactions of PIK-108 in the catalytic site are similar in the
murine WT and human mutant forms, while they are significantly different
in the case of human WT PI3Ka protein. PIK-108 binding in the non-ATP
pocket also differs significantly among the three variants. Finally, we
examine whether the non-ATP binding site is implicated in PI3Ka allostery
in terms of its communication with the active site using principal
component analysis and perform in vitro experiments to verify our
hypotheses.
http://pubs.acs.org/doi/abs/10.1021/jp506423e
