Novel inhibitors of AKT: assessment of a different approach targeting the pleckstrin homology domain.

Protein kinase B/AKT plays a central role in cancer. The serine/threonine kinase is overexpressed or constitutively active in many cancers and has been validated as a therapeutic target for cancer treatment. However, targeting the kinase activity has revealed itself to be a challenge due to non-selectivity of the compounds towards other kinases. This review summarizes other approaches scientists have developed to inhibit the activity and function of AKT. They consist in targeting the pleckstrin homology (PH) domain of AKT. Indeed, upon the generation of 3-phosphorylated phosphatidylinositol phosphates (PI3Ps) by PI3-kinase (PI3K), AKT translocates from the cytosol to the plasma membrane and binds to the PI3Ps via its PH domain. Thus, several analogs of PI3Ps (PI Analogs or PIAs), alkylphospholipids (APLs), such as edelfosine or inositol phophates (IPs) have been described that inhibit the binding of the PH domain to PI3Ps. Recent allostertic inhibitors and small molecules that do not bind the kinase domain but affect the kinase activity of AKT, presumably by interacting with the PH domain, have been also identified. Finally, several drug screening studies spawned novel chemical scaffolds that bind the PH domain of AKT. Together, these approaches have been more or less sucessfull in vitro and to some extent translated in preclinical studies. Several of these new AKT PH domain inhibitors exhibit promising anti-tumor activity in mouse models and some of them show synergy with ionizing radiation and chemotherapy. Early clinical trials have started and results will attest to the validity and efficacy of such approaches in the near future.

Molecular modeling studies of the Akt PH domain and its interaction with phosphoinositides.

The serine-threonine protein kinase Akt is a direct downstream target of phosphatidylinositol 3-kinase (PI3-K). The PI3-K-generated phospholipids regulate Akt activity via directly binding to the Akt PH domain. The binding of PI3-K-generated phospholipids is critical to the relocalization of Akt to the plasma membrane, which plays an important role in the process of Akt activation. Activation of the PI3-K/Akt signaling pathway promotes cell survival. To elucidate the structural basis of the interaction of PI3-K-generated phospholipids with the Akt PH domain with the objective of carrying out structure-based drug design, we modeled the three-dimensional structure of the Akt PH domain. Comparative modeling-based methods were employed, and the modeled Akt structure was used in turn to construct structural models of Akt in complex with selected PI3-K-generated phospholipids using the computational docking approach. The model of the Akt PH domain consists of seven beta-strands forming two antiparallel beta-sheets capped by a C-terminal alpha-helix. The beta1-beta2, beta3-beta4, and beta6-beta7 loops form a positively charged pocket that can accommodate the PI3-K-generated phospholipids in a complementary fashion through specific hydrogen-bonding interactions. The residues Lys14, Arg25, Tyr38, Arg48, and Arg86 form the bottom of the binding pocket and specifically interact with the 3- and 4-phophate groups of the phospholipids, while residues Thr21 and Arg23 are situated at the wall of the binding pocket and bind to the 1-phosphate group. The predicted binding mode is consistent with known site-directed mutagenesis data, which reveal that mutation of these crucial residues leads to the loss of Akt activity. Moreover, our model can be used to predict the binding affinity of PI3-K-generated phospholipids and rationalize the specificity of the Akt PH domain for PI(3,4)P2, as opposed to other phospholipids such as PI(3)P and PI(3,4,5)P3. Taken together, our modeling studies provide an improved understanding of the molecular interactions present between the Akt PH domain and the PI3-K-generated phospholipids, thereby providing a solid structural basis for the design of novel, high-affinity ligands useful in modulating the activity of Akt.

3-Deoxy-3-substituted-D-myo-inositol imidazolyl ether lipid phosphates and carbonate as inhibitors of the phosphatidylinositol 3-kinase pathway and cancer cell growth.

3-Modified D-myo-inositol imidazolyl ether lipid phosphates and a carbonate were synthesized and evaluated as inhibitors of P13-K and Akt. These data are presented along with IC50 values for the inhibition of the growth of three cancer cell lines.

3-(Hydroxymethyl)-bearing phosphatidylinositol ether lipid analogues and carbonate surrogates block PI3-K, Akt, and cancer cell growth.

Phosphatidylinositol 3-kinase (PI3-K) phosphorylates the 3-position of phosphatidylinositol to give rise to three signaling phospholipids. Binding of the pleckstrin homology (PH) domain of Akt to membrane PI(3)P's causes the translocation of Akt to the plasma membrane bringing it into contact with membrane-bound Akt kinase (PDK1 and 2), which phosphorylates and activates Akt. Akt inhibits apoptosis by phosphorylating Bad, thus promoting its binding to and blockade of the activity of the cell survival factor Bcl-x. Herein we present the synthesis and biological activity of several novel phosphatidylinositol analogues and demonstrate the ability of the carbonate group to function as a surrogate for the phosphate moiety. Due to a combination of their PI3-K and Akt inhibitory activities, the PI analogues 2, 3, and 5 proved to be good inhibitors of the growth of various cancer cell lines with IC(50) values in the 1-10 microM range. The enhanced Akt inhibitory activity of the axial hydroxymethyl-bearing analogue 5 compared to its equatorial counterpart 6 is rationalized based upon postulated differences in the H-bonding patterns of these compounds in complex with a homology modeling generated structure of the PH domain of Akt. This work represents the first attempt to examine the effects of 3-modified PI analogues on these two crucial cell signaling proteins, PI3-K and Akt, in an effort to better understand their cell growth inhibitory properties.

Modulation of EGF receptor activity by changes in the GM3 content in a human epidermoid carcinoma cell line, A431.

Gangliosides have been described as modulators of growth factor receptors. For example, GM3 addition in cell culture medium inhibits epidermal growth factor (EGF)-stimulated receptor autophosphorylation. Furthermore, depletion of ganglioside by sialidase gene transfection appeared to increase EGF receptor (EGFR) autophosphorylation. These data suggested that changes in GM3 content may result in different responses to EGF. In this study, the ceramide analog d-threo-1-phenyl-2-decannoylamino-3-morpholino-1-propanol ([D]-PDMP), which inhibits UDP-glucose-ceramide glucosyltransferase, and addition of GM3 to the culture medium were used to study the effects of GM3 on the EGFR. Addition of 10 microM [D]-PDMP to A431 cells resulted in significant GM3 depletion. Additionally, EGFR autophosphorylation was increased after EGF stimulation. When exogenous GM3 was added in combination with [D]-PDMP, the enhanced EGFR autophosphorylation was returned to control levels. [D]-PDMP also increased EGF-induced cell proliferation, consistent with its effect on autophosphorylation. Once again, the addition of GM3 in combination with [D]-PDMP reversed these effects. These results indicate that growth factor receptor functions can be modulated by the level of ganglioside expression in cell lines. Addition of GM3 inhibits EGFR activity and decrease of GM3 levels using [D]-PDMP treatment enhances EGFR activity. Modulation of growth factor receptor function may provide an explanation for how transformation-dependent ganglioside changes contribute to the transformed phenotype.

Metformin modulates insulin receptor signaling in normal and cholesterol-treated human hepatoma cells (HepG2).

The effects of the biguanide anti-hyperglycemic agent, metformin (N,N'-dimethyl-biguanide), on insulin signaling was studied in a human hepatoma cell line (HepG2). Cells were cultured in the absence (control cells) or in the presence of 100 microM of a cholesterol derivative, hemisuccinate of cholesterol. Cholesterol hemisuccinate-treatment alters cholesterol and lipid content of HepG2 and modulates membrane fluidity. Cholesterol hemisuccinate-treatment induces a decrease in insulin responsiveness and creates an 'insulin-resistant' state in these cells. Exposure to 100 microM of metformin resulted in a significant enhancement of insulin-stimulated lipogenesis in control and cholesterol hemisuccinate-treated cells. In control cells, metformin altered glycogenesis in a biphasic manner. In cholesterol hemisuccinate-treated cells, metformin inhibited basal glycogenesis but restored insulin-stimulated glycogenesis. Hence, to understand the mechanism of metformin action, we analyzed early steps in the insulin signaling pathway, including insulin receptor autophosphorylation, mitogen-activated-protein kinase and phosphatidylinositol 3-kinase activities, in both control and cholesterol hemisuccinate-treated cells. Overall, the results suggest that metformin may interact with the insulin receptor and/or a component involved in the early steps of insulin signal transduction.

Incorporation of exogenous lipids modulates insulin signaling in the hepatoma cell line, HepG2.

The lipid content of cultured cells can be experimentally modified by supplementing the culture medium with specific lipids or by the use of phospholipases. In the case of the insulin receptor, these methods have contributed to a better understanding of lipid disorder-related diseases. Previously, our laboratory demonstrated that experimental modification of the cellular lipid composition of an insulin-sensitive rat hepatoma cell line (ZHC) resulted in an alteration in insulin receptor binding and biological action (Bruneau et al., Biochim. Biophys. Acta 928 (1987) 287-296/297-304). In this paper, we have examined the effects of lipid modification in another hepatoma cell line, HepG2. Exogenous linoleic acid (LA, n-6), eicosapentaenoic acid (EPA, n-3) or hemisuccinate of cholesterol (CHS) was added to HepG2 cells, to create a cellular model in which membrane composition was modified. In this model, we have shown that: (1) lipids were incorporated in treated HepG2 cells, but redistributed differently when compared to treated ZHC cells; (2) that insulin signaling events, such as insulin receptor autophosphorylation and the phosphorylation of the major insulin receptor substrate (IRS-1) were altered in response to the addition of membrane lipids or cholesterol derived components; and (3) different lipids affected insulin receptor signaling differently. We have also shown that the loss of insulin receptor autophosphorylation in CHS-treated cells can be correlated with a decreased sensitivity to insulin. Overall, the results suggest that the lipid environment of the insulin receptor may play an important role in insulin signal transduction.

Sialidase gene transfection enhances epidermal growth factor receptor activity in an epidermoid carcinoma cell line, A431.

Glycosphingolipids expressed in cancer cells have been implicated in the modulation of tumor cell growth through their interaction with transmembrane signaling molecules such as growth factor receptors. For glycosphingolipids to interact with growth factor receptors, the presence of sialic acid seems to be essential. Stable transfection of a gene encoding a soluble Mr 42,000 sialidase into a human epidermoid carcinoma cell line (A431) provided an approach by which the level of terminal lipid-bound sialic acid on the cell surface could be altered. In the sialidase-positive clones, the level of ganglioside GM3 was diminished, and little change was observed in protein sialylation. Sialidase-transfected cells grew faster than control cells. Sialidase expression did not modify the binding of epidermal growth factor (EGF) to its receptor but enhanced EGF receptor (EGFR) tyrosine autophosphorylation as compared to that of parental cells or cells transfected with the vector (pcDNA3) alone. Moreover, the phosphorylation of the EGFR, as well as other protein substrates, was observed at low EGF concentrations, suggesting an increase in the receptor kinase sensitivity. These data provided evidence that changes in ganglioside expression in cancer cells by appropriate gene transfection can dramatically affect EGFR kinase activity. Hence, the modulation of ganglioside expression may represent an approach to alter tumor cell growth.

Growth factor receptors as targets for therapy in pediatric brain tumors.

Growth factor receptors (GFRs) have been described as overexpressed in several types of brain tumors. Overexpression of these transmembrane proteins is considered to be an important part of tumorigenesis. Genetic as well as epigenetic modulation of the receptors have to be considered when trying to understand the role of GFRs in tumors or as targets for tumor therapy. GFR function can be modulated by membrane components (e.g. gangliosides) or by the change in receptor glycosylation. These types of changes and the occurrence of the expression of mutated receptor expressed in tumor cell can result in altered signaling. In this review, we have focused on GFRs, their expression and mutations in brain tumors. Recently the correlation between GFR expression and patient outcome has suggested that these tyrosine kinases and their signaling might play a decisive role in the course of patients with brain tumors. The importance of GFRs as possible targets for brain tumor therapy is also discussed.