Journal of Molecular Biology
A Structural Comparison of Inhibitor Binding to PKB, PKA and PKA-PKB Chimera
Introduction
Protein kinase B (PKB/Akt) is a key component of the PI3K kinase pathway, which is responsible for cell-proliferation and survival.1., 2., 3., 4. It acts as a central node on the pathway, phosphorylating a large number of downstream proteins, including GSK3β, FKHRL1, BAD and TSC2, and thereby regulating cell growth, protein translation, apoptosis, and cell-cycle progression.
Numerous hormones, growth and survival factors trigger the activation of PKB via PI3K-mediated generation of the second messenger PtdIns(3,4,5)P3 (PIP3). The interaction of PIP3 with the PH-domain of PKB localises the enzyme to two membrane associated kinases, PDK1 and rictor-mTOR,5., 6. responsible for phosphorylation of PKB on its activation segment and hydrophobic motif, respectively. These two phosphorylation events cooperate to activate PKB some 1000-fold. Down-regulation of PKB is achieved via a combination of PIP3 dephosphorylation by the lipid phosphatase PTEN (thus antagonising PI3K), and PP2A-mediated dephosphorylation of PKB.
PKB is increasingly being recognized as an important therapeutic target for the treatment of malignancy, due to the large number of human cancers in which the PI3K pathway is disregulated.1., 7., 8., 9., 10., 11. Deletion or mutation of the tumour suppressor gene PTEN is found in several common human tumours, and leads to constitutive PKB activity, with continuous high levels of signalling through the PI3K pathway. In addition, mutation and amplification of the PI3K kinase, PIK3CA, and amplification of the three PKB isoforms, have been observed in ovarian, prostate and cervical tumours, amongst others.12., 13. Most recently, somatic mutations of PKBβ have also been linked to colorectal cancers.14
Blockade of PKB-mediated signalling can be achieved by disrupting the phosphorylation, and hence the activation, of the enzyme by inhibition of upstream kinases such as PI3K, mTOR and PDK1, or through inhibiting the association of the enzyme's PH domain with phosphatidylinositols.15., 16., 17., 18. One of the most active area of research, however, is the development of inhibitors that target the ATP-site of the PKB catalytic domain, and several chemical series are currently in pre-clinical development.19., 20., 21., 22., 23., 24., 25.
In 2002, the three-dimensional structure of active, phosphorylated PKBβ (Akt-2) was solved26 (in complex with the nucleotide analogue AMP-PNP), and has given important insights into the design of novel inhibitors targeted towards the ATP-binding site. However, to date, all detailed structure-based accounts of PKB-inhibitor development described in the literature have employed the closely related kinase, PKA, as a surrogate system due to its facile co-crystallization and soakability with a range of ligands.19., 20., 22., 23., 24., 25., 26., 27., 28. PKA is highly homologous to PKB, sharing approximately 45% sequence identity with the kinase domain, and this rises to approximately 80% within the ATP site, with only three key amino acid differences within the cleft itself.
This use of PKA, either as wild-type,20., 22., 23., 24., 25. or as a mutant PKA, in which ATP-site residues that differ from PKB are mutated to form a “PKA-PKB chimera”,19., 27. has provided a useful approach, and allowed the development of inhibitors with low nanomolar potency. However, there is a clear requirement to validate the general applicability of such surrogate systems, particularly for the development of molecules with a PKB versus PKA selective profile. A comparison with PKB-ligand structural information would allow such an analysis to be carried out, and would also give a more rigorous and complete understanding of the molecular recognition with this important enzyme.
Despite extensive screening for novel conditions, neither apo-crystallization of PKBβ nor co-crystallization with inhibitors other than AMP-PNP was successful. We have therefore developed a “back-soaking” method for obtaining high resolution, phosphorylated PKBβ-inhibitor crystal structures, which has allowed us to fully exploit PKB structural information within the drug discovery process. To illustrate the use of this method, we present the structures of two contrasting inhibitors (Figure 1) bound to PKB, and provide a comparison of their binding modes with both PKA and PKA-PKB. The isoquinoline sulphonamide inhibitor 1 is derived from a series that exhibits little or no PKB/PKA selectivity (IC50(PKA) = 0.17 μM, IC50(PKBβ) = 0.23 μM25), whereas the indazole-based inhibitor 2 (A-443654) exhibits approximately 40-fold selectivity for PKB ((Ki(PKA) = 6.3 nM, Ki(PKBα) = 160 pM24). This use of PKB-inhibitor crystallography allows us to present a detailed comparison of the binding of selective and non-selective inhibitors in each system for the first time.
Section snippets
PKB-inhibitor structural overview and comparison with PKA
PKBβ-inhibitor co-crystals with compounds 1 and 2 were obtained using a back-soaking protocol (see Materials and Methods), X-ray diffraction data were collected, and the structures were refined to 1.8 Å and 2.3 Å resolution, respectively. The overall protein structure is in each case essentially identical to that observed for the published structure of activated PKB in complex with the nucleotide analogue AMP-PNP.26 PKB adopts the classical bi-lobal kinase fold, with a predominantly β-sheet
Discussion
Over the last few years, PKB has increasingly become recognized as a potential target for the treatment of malignancy due to its central position in a network of biochemical pathways that regulate cellular growth and proliferation. The determination of the structure of activated PKBβ complexed with AMP-PNP was an important step towards using a structure-based approach to PKB inhibitor development, but design studies presented in the literature have until now utilized the experimentally more
PKA crystallography
The alpha catalytic subunit of bovine PKA was expressed, purified and crystallized with reference to previously described protocols.28., 43. Hanging drops containing 17 mg ml−1 of tetra-phospho PKA, 25 mM Mes-BisTris (pH 6.5), 75 mM LiCl, 0.1 mM EDTA, 1 mM DTT, 1.5 mM octanoyl-N-methylglucamide and 1 mM PKI(5-24) were equilibrated at 4 °C against 15% (v/v) methanol. Crystals of apo PKA appeared overnight, and were soaked for approximately 18 h at 4 °C in a solution containing 10% (w/v) PEG 400,
Acknowledgements
The authors acknowledge Andrew Sharff for assistance with PKB protein purification, Wendy Blakemore for assistance with the PKA-PKB chimera crystallography and Lisa Seavers for the bioassay results. This work was funded in part by Cancer Research UK [CUK] Programme Grant C309/A2187.
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