Novel Inositol Phospholipid Headgroup Surrogate Crystallized in the Pleckstrin Homology Domain of Protein Kinase Bα

Abstract

Protein kinase B (PKB/Akt) plays a key role in cell signaling. The PH domain of PKB binds phosphatidylinositol 3,4,5-trisphosphate translocating PKB to the plasma membrane for activation by 3-phosphoinositide-dependent protein kinase 1. The crystal structure of the headgroup inositol 1,3,4,5-tetrakisphosphate Ins(1,3,4,5)P 4 -PKB complex facilitates in silico ligand design. The novel achiral analogue benzene 1,2,3,4-tetrakisphosphate (Bz(1,2,3,4)P 4 ) possesses phosphate regiochemistry different from that of Ins(1,3,4,5)P 4 and surprisingly binds with similar affinity as the natural headgroup. Bz(1,2,3,4)P 4 co-crystallizes with the PKB␣ PH domain in a fashion also predictable in silico. The 2-phosphate of Bz(1,2,3,4)P 4 does not interact with any residue, and the D5-phosphate of Ins(1,3,4,5)P 4 is not mimicked by Bz(1,2,3,4)P 4 . Bz(1,2,3,4)P 4 is an example of a simple inositol phosphate surrogate crystallized in a protein, and this approach could be applied to design modulators of inositol polyphosphate binding proteins.

A novel inhibitor of the PI3K/Akt pathway based on the structure of inositol 1,3,4,5,6-pentakisphosphate

“…Indeed we reported that inositol 1,3,4,5,6-pentakisphosphate (InsP 5 ) specifically blocks Akt activation and possesses pro-apoptotic (Razzini et al, 2000;Piccolo et al, 2004), anti-angiogenic and anti-tumour activity in vivo (Maffucci et al, 2005). In addition, some of us recently demonstrated the targeting of the Akt PH domain with an unusual inositol polyphosphate mimic (Mills et al, 2007). Other phosphatidylinositol-based Akt inhibitors also act by inhibiting Akt targeting to the plasma membrane, including ether lipid analogues and PH domain-targeting inhibitors (Kozikowski et al, 2003;Gills et al, 2006;Crowell et al, 2007) such as perifosine, the most developed Akt inhibitor currently available (Kondapaka et al, 2003).…”

Benzene Polyphosphates as Tools for Cell Signalling: Inhibition of Inositol 1,4,5‐Trisphosphate 5‐Phosphatase and Interaction with the PH Domain of Protein Kinase Bα

“…Benzene 1,2,3,4-tetraA C H T U N G T R E N N U N G kis-A C H T U N G T R E N N U N G phosphate (3) [13] and biphenyl 2,3',4,5',6-pentakisphosphate (4) [14] have been used in other studies and were synthesised by published procedures.…”

“…Benzene 1,2-bisphosphate (7), benzene 1,2,3-trisphosphate (10) and benzene 1,3,5-trisphosphate (13) were synthesised by phosphitylation of the appropriate polyphenols with diethyl chlorophosphite to give the corresponding bis-or trisphosphites (Scheme 1). The intermediates were oxidised with mchloroperoxybenzoic acid (mCPBA) to afford the corresponding polyphosphorylated derivatives (6, 9 and 12) in approximately 50 % yield for each compound.…”

“…[13] Interestingly, the lipid analogue diC 8 , also a molecule of the benzene phosphate class, consists of two phosphorylated benzene rings that, from modelling studies, appear to interact with more sites on PKBa PH domain than any other compound, resulting in a potent binding affinity (K i = 27 nm), 8.5 times higher than that of diC 8 PtdInsA C H T U N G T R E N N U N G (3,4,5)P 3 . The binding affinity for BzA C H T U N G T R E N N U N G (1,3,5)P 3 13 (K i = 2.18 mm) is interesting in the context of 4, since one ring is functionalised with the same benzene 1,3,5-trisphosphate motif (2,4,6-trisphosphate of 4) and the adjacent ring has a benzene 1,3-bisphosphate motif (3',5'-bis-A C H T U N G T R E N N U N G phosphate of 4).…”

“…[13] Protein kinase B (PKB, also known as Akt) is an enzyme involved in inositol lipid signalling and is made up of three distinct, highly conserved enzymes: PKBa, PKBb and PKBg (also known as Akt1, Akt2 and Akt3), which have their own functions within cells. [17][18][19] A pleckstrin homology domain (PH domain) binds phosphatidylinositol 3,4,5-trisphosphate [PtdIns-A C H T U N G T R E N N U N G (3,4,5)P 3 ], which is required for PKB phosphorylation and activation.…”