An organometallic protein kinase inhibitor pharmacologically activates p53 and induces apoptosis in human melanoma cells.

Unlike other tumors, melanomas harbor wild-type (WT) p53 but exhibit impaired p53-dependent apoptosis. The mechanisms for the impaired p53 activation are poorly understood but may be linked to the high expression of the p53 suppressor Mdm2, which is found in >50% of melanoma lesions. Here, we describe an organometallic glycogen synthase kinase 3beta (GSK3beta) inhibitor (DW1/2) as a potent activator of p53 and inducer of cell death in otherwise highly chemoresistant melanoma cells. Using RNA interference and pharmacologic approaches, we show that p53 is required for the cytotoxic effects of this organometallic inhibitor. The DW1/2 compound was barely able to induce cell death in melanoma cells with p53 mutations, further confirming the requirement for p53-WT in the cytotoxic effects of the GSK3beta inhibition. Mechanistic analysis of the p53-dependent cell death indicated an apoptotic mechanism involving depolarization of mitochondrial membrane potential, caspase cleavage, and elevated NOXA expression. The effect of p53 was not simply due to passive up-regulation of protein expression as adenoviral-mediated overexpression of p53 was not able to induce cell death. Treatment of melanoma cells with DW1/2 was instead found to decrease levels of Mdm2 and Mdm4. The importance of Mdm2 down-regulation in DW1/2-induced apoptosis was confirmed by treating the p53-WT cells with the p53/Mdm2 antagonist Nutlin-3. Taken together, our data provide a new strategy for the pharmacologic activation of p53 in melanoma, which may be a viable approach for overcoming apoptotic resistance in melanoma and offer new hope for rational melanoma therapy.

Structure-based design of an organoruthenium phosphatidyl-inositol-3-kinase inhibitor reveals a switch governing lipid kinase potency and selectivity.

Mutations that constitutively activate the phosphatidyl-inositol-3-kinase (PI3K) signaling pathway, including alterations in PI3K, PTEN, and AKT, are found in a variety of human cancers, implicating the PI3K lipid kinase as an attractive target for the development of therapeutic agents to treat cancer and other related diseases. In this study, we report on the combination of a novel organometallic kinase inhibitor scaffold with structure-based design to develop a PI3K inhibitor, called E5E2, with an IC 50 potency in the mid-low-nanomolar range and selectivity against a panel of protein kinases. We also show that E5E2 inhibits phospho-AKT in human melanoma cells and leads to growth inhibition. Consistent with a role for the PI3K pathway in tumor cell invasion, E5E2 treatment also inhibits the migration of melanoma cells in a 3D spheroid assay. The structure of the PI3Kgamma/E5E2 complex reveals the molecular features that give rise to this potency and selectivity toward lipid kinases with implications for the design of a subsequent generation of PI3K-isoform-specific organometallic inhibitors.

Similar biological activities of two isostructural ruthenium and osmium complexes.

In this study, we probe and verify the concept of designing unreactive bioactive metal complexes, in which the metal possesses a purely structural function, by investigating the consequences of replacing ruthenium in a bioactive half-sandwich kinase inhibitor scaffold by its heavier congener osmium. The two isostructural complexes are compared with respect to their anticancer properties in 1205 Lu melanoma cells, activation of the Wnt signaling pathway, IC(50) values against the protein kinases GSK-3beta and Pim-1, and binding modes to the protein kinase Pim-1 by protein crystallography. It was found that the two congeners display almost indistinguishable biological activities, which can be explained by their nearly identical three-dimensional structures and their identical mode of action as protein kinase inhibitors. This is a unique example in which the replacement of a metal in an anticancer scaffold by its heavier homologue does not alter its biological activity.

Platinum complex as a nanomolar protein kinase inhibitor.

A pyridocarbazole platinum complex, which matches the overall shape of the natural product staurosporine, binds with high affinity at the adenosine triphosphate binding site of glycogen synthase kinase 3 (GSK-3alpha).

Ruthenium half-sandwich complexes as protein kinase inhibitors: derivatization of the pyridocarbazole pharmacophore ligand.

A general route to ruthenium pyridocarbazole half-sandwich complexes is presented and applied to the synthesis of sixteen new compounds, many of which have modulated protein kinase inhibition properties. For example, the incorporation of a fluorine into the pyridine moiety increases the binding affinity for glycogen synthase kinase 3 by almost one order of magnitude. These data are supplemented with cyclic voltammetry experiments and a protein co-crystallographic study.

Organometallic compounds with biological activity: a very selective and highly potent cellular inhibitor for glycogen synthase kinase 3.

A chiral second-generation organoruthenium half-sandwich compound is disclosed that shows a remarkable selectivity and cellular potency for the inhibition of glycogen synthase kinase 3 (GSK-3). The selectivity was evaluated against a panel of 57 protein kinases, in which no other kinase was inhibited to the same extent, with a selectivity window of at least tenfold to more than 1000-fold at 100 microM ATP. Furthermore, a comparison with organic GSK-3 inhibitors demonstrated the superior cellular activity of this ruthenium compound: wnt signaling was fully induced at concentrations down to 30 nM. For comparison, the well-established organic GSK-3 inhibitors 6-bromoindirubin-3'-oxime (BIO) and kenpaullone activate the wnt pathway at concentrations that are higher by around 30-fold and 100-fold, respectively. The treatment of zebrafish embryos with the organometallic inhibitor resulted in a phenotype that is typical for the inhibition of GSK-3. No phenotypic change was observed with the mirror-imaged ruthenium complex. The latter does not, in fact, show any of the pharmacological properties for the inhibition of GSK-3. Overall, these results demonstrate the potential usefulness of organometallic compounds as molecular probes in cultured cells and whole organisms.

An organometallic inhibitor for glycogen synthase kinase 3.

Replacing natural products with kinetically inert metal complexes may lead to a new class of therapeutics in which a metal center plays the role of an innocent bystander, organizing the orientation of the organic ligands in the receptor space. As an example of this approach, a ruthenium complex is described that copies the binding mode of indolocarbazole protein kinase inhibitors and serves as a reversible, low-nanomolar inhibitor for glycogen synthase kinase 3 (GSK-3).