A diverse range of cellular functions are influenced by the activity of protein kinase B (PKB, also known as Akt). PKB is involved in a number of physiological pathways, perturbation of which can lead to pathological conditions. PKB activation has been shown to involve binding of its pleckstrin homology (PH) domain to PtdIns(3,4)P2 and PtdIns(3,4,5)P3 generated on the inner face of the plasma membrane upon receipt of an external hormonal signal.
To study PKB independently of other inositol-phospholipid binding proteins which recognise the same lipid signals, their mutual dependence on the same ligand must be broken. It is proposed that this can be achieved by mutating PKB‟s PtdInsPn binding PH domain to accept an unnatural ligand. By altering the inositol-phospholipid head-group to include an additional carboxylic acid substituent, its binding to the wild-type PH domains should be inhibited through steric compression. Disruption of a salt-bridge in the wall of the binding pocket by point mutation of glutamate 17 to alanine, E17A, introduces an additional positively charged pocket into the PH domain of PKB, which should bind with the tailored lipid, resulting in a polar group exchange receptor-ligand pair.
To this end, a novel 2-O-acetic acid functionalised analogue of Ins(1,3,4,5)P4 was synthesised. The key steps were the protection/deprotection of myo-inositol‟s hydroxyls, enabling selective functionalisation of the 2-hydroxyl, and phosphorylation of the 1,3,4,5-O positions. Using an allyl ether as a masked form of acetic acid allowed a divergent synthetic strategy to be employed to access additional 2-O analogues.
Both the wild-type and the E17A mutant of the PH domain were expressed in order to assess ligand binding, and determine whether an ion pair exchange receptor-ligand interaction occurred. Intrinsic tryptophan fluorescence spectrometry was initially used, but was unsuccessful due to denaturing problems arising from isolating the protein. Isothermal titration calorimetry suggested binding, but protein concentration could not be increased high enough for the binding energy to become significantly greater than the energy of mixing.
The analogues were also tested as potential inositol poly-phosphate phosphatase catalytic (IPPc) domain inhibitors.