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 3B (GSK3B) 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 GSK3B 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 adenoviralmediated 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.

Keeping in shape with half a sandwich: The structure of a picomolar organoruthenium inhibitor bound to the ATP‐binding site of the protein kinase Pim‐1 (see picture) demonstrates that the ruthenium center has solely a structural role in organizing the organic ligands in the three‐dimensional receptor space, thus yielding a structure that is complementary in shape and functional group presentation to the active site of Pim‐1.

Complementing organic elements with a metal center provides new opportunities for building three-dimensional structures with unique and defined shapes. Such access to unexplored chemical space may lead to the discovery of molecules with unprecedented properties. Along these lines, this account article describes our successful design of highly potent and selective rutheniumbased inhibitors for the protein kinases GSK-3 and Pim-1 by using the class of indolocarbazole alkaloids as a lead structure. The described ruthenium complexes are kinetically inert scaffolds in which the ruthenium has the function to organize the orientation of the organic ligands in the three-dimensional space.

The vast majority of specific enzyme inhibitors are small organic molecules that gain their specificity by a combination of weak interactions, including hydrogen bonding, electrostatic contacts, and hydrophobic interactions. In contrast, inorganic compounds find applications in medicinal chemistry predominately for their reactivity and imaging properties. 1 We started a research program that aims in exploring the versatility of organometallic and inorganic compounds as structural scaffolds for the design of specific enzyme inhibitors. 2,3 It is noteworthy that coordinative bonds with transition metals such as ruthenium can reach kinetic stabilities that are comparable with those of covalent bonds. 4 With this in mind, a ruthenium center may be considered as a virtual "hypervalent carbon" with unique structural opportunities.We recently introduced a strategy for developing ruthenium complexes that target the ATP-binding site of protein kinases by copying structural features of small organic molecule inhibitors. 5 The adenine base of ATP is lined with a cleft-forming set of conserved hydrophobic residues and forms two hydrogen bonds to the backbone of the hinge between the N-terminal and C-terminal domain. 6 Small-molecule inhibitors usually copy this binding mode. 7 For example, the protein kinase inhibitor staurosporine 1 contains the planar hydrophobic indolo[2,3-a]carbazole aglycon 2a in which the lactam moiety mimics the hydrogen bonding pattern of the adenine base (Figure 1). 8 We envisioned that replacing the indolocarbazole alkaloid scaffold with metal complexes in which the structural features of the indolocarbazole aglycon 2a or the related arcyriaflavin A 2b are retained in one of the ligands would allow metal complexes to be targeted to the ATP-binding site of protein kinases. Potent and specific inhibitors for a particular kinase could then be obtained by assembling elaborate structures around the metal center. As a demonstration of this concept, we here report the organometallic ruthenium compound 3 as an extremely potent inhibitor for the glycogen synthase kinase 3 (GSK-3).The key component of our design is the novel pyridocarbazole ligand 4, derived from arcyriaflavin A 2b by just replacing one indole moiety with a pyridine (shown in red in Figure 1). An X-ray structure of the N-benzylated derivative of 3 proves that ligand 4 can in fact serve as a bidentate ligand for ruthenium, having one classical coordinative bond with the pyridine (Ru1-N19 ) 2.13 Å) in addition to one covalent σ-bond with the indole nitrogen (Ru1-N2 ) 2.11 Å) (indicated in green in Figure 1). 9 The coordination sphere is further filled up by a cyclopentadienyl and CO group. This neutral half-sandwich ruthenium complex 3 is stable under air and in water and can even withstand the presence of millimolar concentrations of thiols as determined by 1 H NMR spectroscopy.Screening a small library of ruthenium complexes against a panel of protein kinases identified 3 as an extremely potent inhibitor for GSK-3. The concentration at ...

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.