Howard Bregman

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.

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 μM 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.

The crystal structure of BRAF in complex with an organoruthenium inhibitor reveals a mechanism for inhibition of an active form of BRAF kinase.

Substitution mutations in the BRAF serine/threonine kinase are found in a variety of human cancers. Such mutations occur in approximately 70% of human malignant melanomas, and a single hyperactivating V600E mutation is found in the activation segment of the kinase domain and accounts for more than 90% of these mutations. Given this correlation, the molecular mechanism for BRAF regulation as well as oncogenic activation has attracted considerable interest, and activated forms of BRAF, such as BRAF(V600E), have become attractive targets for small molecule inhibition. Here we report on the identification and subsequent optimization of a potent BRAF inhibitor, CS292, based on an organometallic kinase inhibitor scaffold. A cocrystal structure of CS292 in complex with the BRAF kinase domain reveals that CS292 binds to the ATP binding pocket of the kinase and is an ATP competitive inhibitor. The structure of the kinase-inhibitor complex also demonstrates that CS292 binds to BRAF in an active conformation and suggests a mechanism for regulation of BRAF by phosphorylation and BRAF(V600E) oncogene-induced activation. The structure of CS292 bound to the active form of the BRAF kinase also provides a novel scaffold for the design of BRAF(V600E) oncogene selective BRAF inhibitors for therapeutic application.

Crystal Structure of the PIM2 Kinase in Complex with an Organoruthenium Inhibitor

BACKGROUND The serine/threonine kinase PIM2 is highly expressed in human leukemia and lymphomas and has been shown to positively regulate survival and proliferation of tumor cells. Its diverse ATP site makes PIM2 a promising target for the development of anticancer agents. To date our knowledge of catalytic domain structures of the PIM kinase family is limited to PIM1 which has been extensively studied and which shares about 50% sequence identity with PIM2. PRINCIPAL FINDINGS Here we determined the crystal structure of PIM2 in complex with an organoruthenium complex (inhibition in sub-nanomolar level). Due to its extraordinary shape complementarity this stable organometallic compound is a highly potent inhibitor of PIM kinases. SIGNIFICANCE The structure of PIM2 revealed several differences to PIM1 which may be explored further to generate isoform selective inhibitors. It has also demonstrated how an organometallic inhibitor can be adapted to the binding site of protein kinases to generate highly potent inhibitors. ENHANCED VERSION This article can also be viewed as an enhanced version in which the text of the article is integrated with interactive 3D representations and animated transitions. Please note that a web plugin is required to access this enhanced functionality. Instructions for the installation and use of the web plugin are available in Text S1.

From imide to lactam metallo-pyridocarbazoles: distinct scaffolds for the design of selective protein kinase inhibitors.

Organometallic pyridocarbazole scaffolds are investigated as protein kinase inhibitors. Whereas our previous designs employed solely a maleimide pharmacophore for achieving the two crucial canonical hydrogen bonds to the hinge region of the ATP binding site, we have now extended our investigations to include the related lactam metallo-pyridocarbazoles. The synthetic access of the two regioisomeric lactam pyridocarbazoles is described, and the distinct biological properties of the two lactam scaffolds are revealed by employing a ruthenium half sandwich complex as a model system, resulting in organometallic lead structures for the inhibition of the protein kinases TrkA and CLK2. These new lactam metallo-pyridocarbazoles expand our existing molecular toolbox and assist toward the generation of metal complex scaffolds as lead structures for the design of selective inhibitors for numerous kinases of the human kinome.