Nicholas Pagano

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.

Toward the Development of a Potent and Selective Organoruthenium Mammalian Sterile 20 Kinase Inhibitor

Mammalian sterile 20 (MST1) kinase, a member of the sterile 20 (Ste-20) family of proteins, is a proapoptotic cytosolic kinase that plays an important role in the cellular response to oxidative stress. In this study, we report on the development of a potent and selective MST1 kinase inhibitor based on a ruthenium half-sandwich scaffold. We show that the enantiopure organoruthenium inhibitor, 9E1, has an IC50 value of 45 nM for MST1 and a greater than 25-fold inhibitor selectivity over the related Ste-20 kinases, p21 activated kinase 1 (PAK1), and p21 activated kinase 4 (PAK4) and an almost 10-fold selectivity over the related thousand-and-one amino acids kinase 2 (TAO2). Compound 9E1 also displays a promising selectivity profile against unrelated protein kinases; however, the proto-oncogene serine/threonine protein kinase PIM1 (PIM-1) and glycogen synthase kinase 3 (GSK-3beta) are inhibited with IC50 values in the low nanomolar range. We also show that 9E1 can inhibit MST1 function in cells. A cocrystal structure of a related compound with PIM-1 and a homology model with MST1 reveals the binding mode of this scaffold to MST1 and provides a starting point for the development of improved MST1 kinase inhibitors for possible therapeutic application.

Extremely Tight Binding of a Ruthenium Complex to Glycogen Synthase Kinase 3

Pharmaceutical industry and chemical biology are dominated by organic chemistry with inorganic compounds playing only a minor role. This is well illustrated by a review of FDA approved drugs during 2007 in which not a single compound contains a metal atom, with most compounds being reversible enzyme inhibitors.[1] However, our laboratory recently demonstrated that chemically inert metal complexes can serve as promising scaffolds for the design of enzyme inhibitors and we reported several compounds with high affinities and promising selectivity profiles for protein kinases and lipid kinases.[2–4] For example, we have recently introduced the ruthenium half-sandwich complexes HB12 and DW12 as potent protein kinase inhibitors, in particular for GSK-3 and Pim-1.[5–7] DW12 and its derivatives induce strong biological responses such as the activation of the wnt signaling pathway in mammalian cells, strong pharmacological effects during the development of frog embryos, and the efficient induction of apoptosis in some melanoma cell lines.[8,9] Moreover, in an independent previous study we discovered by a combinatorial approach that the introduction of a D-alanine amide side chain into the η5-cyclopentadienyl moiety of HB12 increased affinity by 40-fold ((RRu)-HB1229).[11,12] Based on these results, we were curious to investigate by how much we could further improve potency if we would combine these beneficial modifications at the cyclopentadienyl and pyridocarbazole moiety in one molecule. Accordingly, we synthesized the individual stereoisomers of NP549 (see supporting information for synthetic details) and found (RRu)-NP549 to be an extremely potent inhibitor for GSK-3β with an IC50 of 40 pM at 100 μM ATP.[13,14] Since this IC50 was measured in presence of the lowest possible GSK-3β concentration of 100 pM, this value reflects an upper limit. Considering that GSK-3β displays a Km for ATP of 15 μM, the binding constant can be estimated to Ki ≤ 5 pM by applying the Cheng-Prusoff equation.[15] With this, (RRu)-NP549 is one of the highest affinity ligands for a protein kinase known to date.[16] In order to investigate the binding mode of this class of organoruthenium complexes to GSK-3β, we crystallized full-length human GSK-3β, soaked it with a solution of enantiomerically pure (RRu)-NP549 and solved to a resolution of 2.4 A (Table 1). The global structure reveals the typical two-lobe protein kinase architecture, connected by a hinge region, with the catalytic domain positioned in a deep intervening cleft and (RRu)-NP549 occupying the ATP-binding site, similar to the binding of staurosporine and synthetic organic inhibitors (Figure 2).[17] Figure 2 Crystal structure of GSK-3β with the ruthenium compound (RRu)-NP549 bound to the ATP-binding site. A) Overview of the complete structure. B) Electron density of the ruthenium complex contoured at 1σ. C) Fit of (RRu)-NP549 into the active ... Table 1 Crystallographic data and refinement statistics. (RRu)-NP549 forms a number of hydrogen bonds within the ATP-binding site of GSK-3β (Figure 3). The maleimide moiety and the indole OH-group establish together three important hydrogen bonds to the backbone of the hinge region: one between the imide NH group and the backbone carbonyl oxygen of Asp133, a second between one of the imide carbonyl groups and the backbone NH of Val135 and the third between the backbone carbonyl oxygen of Val135 and the indole OH. The second carbonyl group of the maleimide moiety forms a water-mediated contact to Asp200. An additional hydrogen bond is established with the amide carbonyl group at the cyclopentadienyl moiety which is in a water-mediated contact to Thr138. The carboxylate group does not form any particular hydrogen bond but is nicely placed close to a positively charged patch formed from Arg141 and Arg144 and thus contributing to electrostatic attraction. Furthermore, the fluoride atom is at a close distance to the amino group of Lys85 (3.1 A) which suggests a weak F…H-N hydrogen bond. Figure 3 Interactions of (RRu)-NP549 within the ATP-binding site of GSK-3β. A) Hydrogen bonding interactions. B) The most important hydrophobic interactions. C) Highlighting the close contact of the CO ligand of (RRu)-NP549 with Gly63 and the small hydrophobic ... (RRu)-NP549 is involved in extensive van der Waals contacts with GSK-3β. A hydrophobic pocket for the pyridocarbazole moiety is built by side chains from more than 10 amino acids, in particular Phe67, Val70, Ala83, Val110, Leu132, Tyr134, Val135, Leu188, and Cys199. Phe67 also packs against the CO ligand and one edge of the cyclopentadienyl moiety, whereas Gln185 interacts with one edge and the face of the cyclopentadienyl ring and the adjacent amide carbonyl group. Finally, the methyl group of the cyclopentadienyl amide side chain forms a hydrophobic contact with the CH2-group of Gly63 within the glycine-rich loop. Most interestingly, the CO ligand comes in particularly close contact to Gly63, with a distance to the methylene group of only 3.1 A. This is below the van der Waals distance and suggests dipolar interactions.[18] We have observed this close contact to the glycine-rich loop also in crystal structures of related organometallic compounds with the protein kinase Pim-1.[7,10] In addition, Gly63, together with the sidechains of Ile62, Val70, and Phe67 create a small hydrophobic pocket in which the CO ligand is buried (Figure 3C). It is noteworthy that replacing the CO by any other monodentate ligand reduces the binding affinity significantly.[19] For example, exchanging the CO group in HB12 against PF3 (CS44) increases the IC50 by around 25-fold, presumably because the PF3 ligand is too big for this pocket, whereas replacing the (η5-C5H5)RuCO moiety in HB12 by the highly similar (η6-C6H6)RuCN fragment (NP930) leads to a diminished affinity by 75-fold (Scheme 1). Such a dramatic effect by replacing a CO ligand with a cyanide we have observed before in a related octahedral scaffold.[19] Although isoelectronic, coordinated CO is hydrophobic,[20,21] whereas coordinated cyanide tends to form hydrogen bonds with its nitrogen lone pair and will therefore not have any desire to bind into the hydrophobic pocket build by the glycine-rich loop.[22,23] These examples demonstrate the importance of the CO group and, in fact, we have yet to find a highly potent and selective ruthenium complex for GSK-3 that lacks this apparently crucial CO ligand. Scheme 1 Ruthenium complex HB12 as a lead scaffold for the design of highly potent GSK-3 inhibitors. NP930 and CS44 are only weak inhibitors for GSK-3. IC50 values were measured at 100 μM ATP. Compounds are racemic if not indicated otherwise. Finally, we compared the relative binding position of (RRu)-NP549 with cocrystal structures of small organic molecules bound to GSK-3β. A superimposition of all available structures demonstrates that (RRu)-NP549 occupies the same area of the ATP-binding site. However, it seems that the position of the CO ligand together with the perpendicular orientation to the pyridocarbazole heterocycle is a unique feature of (RRu)-NP549 which allows Val70 to reach down to the pyridocarbazole moiety, thus maximizing the hydrophobic interactions with the pyridocarbazole moiety and creating the hydrophobic pocket for the CO ligand. Although the pyrane oxygen atom of staurosporine occupies a similar position in the active site compared to the CO oxygen of the ruthenium complex, the glycine-rich loop is in a significantly more open position as displayed in Figure 2E and does not allow the same closure of the active site with its optimized contacts. In conclusion, we here reported an extremely high affinity GSK-3 inhibitor and its binding to the ATP-binding site of GSK-3β. Overall, (RRu)-NP549 perfectly complements the shape of the ATP-binding site and forms three direct hydrogen bonds, two water mediated hydrogen bonds, one fluorine-mediated hydrogen bond, undergoes electrostatic contacts between the carboxylate tail and two arginines, and is involved in van der Waals interactions with more than 10 amino acids. Furthermore, the CO ligand stacks against the glycine-rich loop and is buried in a small pocket which appears to be crucial for affinity and selectivity for GSK-3β. With a Ki value of around 5 pM or less, (RRu)-NP549 is one of the most potent protein kinase inhibitors reported to date and by almost 4 orders of magnitude more potent than the related natural product staurosporine (IC50 = 180 nM at 100 μM ATP), demonstrating that this organoruthenium structure is a priviledged scaffold for the design of GSK-3 inhibitors.

GSK3β inhibition blocks melanoma cell/host interactions by downregulating N-cadherin expression and decreasing FAK phosphorylation.

This study addresses the role of glycogen synthase kinase (GSK)-3β signaling in the tumorigenic behavior of melanoma. Immunohistochemical staining revealed GSK3β to be focally expressed in the invasive portions of 12% and 33% of primary and metastatic melanomas, respectively. GSK3 inhibitors and siRNA knockdown of GSK3β were found to inhibit the motile behavior of melanoma cells in scratch wound, 3D collagen implanted spheroid and modified Boyden chamber assays. Functionally, inhibition of GSK3β signaling was found to suppress N-cadherin expression at the mRNA and protein levels and was associated with decreased expression of the transcription factor Slug. Pharmacological and genetic ablation of GSK3β signaling inhibited the adhesion of melanoma cells to both endothelial cells and fibroblasts and prevented transendothelial migration, an effect rescued by the forced overexpression of N-cadherin. A further role for GSK3β signaling in invasion was suggested by the ability of GSK3β inhibitors and siRNA knockdown to block phosphorylation of FAK and increase the size of focal adhesions. In summary, we have demonstrated a previously unreported role for GSK3β in modulating the motile and invasive behavior of melanoma cells through N-cadherin and FAK. These studies suggest the potential therapeutic utility of inhibiting GSK3β in defined subsets of melanoma.

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.