Jasna Maksimoska

Targeting large kinase active site with rigid, bulky octahedral ruthenium complexes.

A strategy for targeting protein kinases with large ATP-binding sites by using bulky and rigid octahedral ruthenium complexes as structural scaffolds is presented. A highly potent and selective GSK3 and Pim1 half-sandwich complex NP309 was successfully converted into a PAK1 inhibitor by making use of the large octahedral compounds Lambda-FL172 and Lambda-FL411 in which the cyclopentadienyl moiety of NP309 is replaced by a chloride and sterically demanding diimine ligands. A 1.65 A cocrystal structure of PAK1 with Lambda-FL172 reveals how the large coordination sphere of the ruthenium complex matches the size of the active site and serves as a yardstick to discriminate between otherwise closely related binding sites.

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.

The art of filling protein pockets efficiently with octahedral metal complexes.

Complicated natural products, with their structures and properties evolved over millions of years, frequently display specific biological modes of action which can often be traced back to their highly preorganized three dimensional structures that perfectly complement the shape and functional group presentation of their target protein pockets.[1] A recent study analyzed the protein binding properties of compounds from natural as well as synthetic sources and found that protein binding selectivity correlated with shape complexity (defined as the relative content of sp3-hybridized carbons) and stereochemical complexity (defined as the relative content of stereogenic carbons).[2] Octahedral metal complexes may offer an attractive alternative strategy to sophisticated globular and rigid structural templates. They are constructed from a powerful single metal stereocenter with chelating ligands limiting the degree of conformational flexibility, thus achieving “natural-product-like” structural complexities and strinkingly high target specificities as demonstrated by us in several previous studies.[3–6] An important aspect regarding the design of such metal-templated protein binders –which has not been articulated in the past– is the globular space requirement of an octahedral center. The latter significantly increases the demand for a proper design, mainly because the metal must be located at a specific position within the active site in order to be useful. For example, if the metal is located too far within the active site or too close to the protein backbone there will not be enough space available to accommodate the space-demanding octahedral coordination sphere, whereas if the metal is located too far towards the solvent, the metal center cannot easily impact on binding affinity and selectivity. A clear indicator for an advantageous metal position within the protein pocket is a strong influence of the metal coordination sphere on binding affinity and selectivity. We have identified such a priviledged position of the metal within the ATP-binding site of protein kinases by using the now well established staurosporine-inspired metallo-pyridocarbazole scaffold.[3–7] For example, the octahedral organoruthenium complex Λ-FL172 was designed as a selective inhibitor for the p21-activated kinase 1 (PAK1)[8] in which the bidentate pyridocarbazole ligand of the ruthenium complex occupies the adenine pocket.[4] By interacting with the so-called hinge region it places the ruthenium center at a defined position within the ribose binding site, where the additional CO, chloride, and bidentate iminopyridine ligands can form important contacts with other parts of the active site and thereby strongly contribute to binding affinity and selectivity (Figure 1).[4,6] In order to better understand the design of metal-based enzyme inhibitors, we wondered if this design is unique or whether other scaffolds with metals located at other positions within the active site could yield similar or even better results. To address this question, we designed new scaffolds[9] and uncovered a simple ruthenium complex (R)-1 which places the metal at a distinct position within the ATP-binding site, contains a completely different set of coordinating ligands, but at the same time shows an improved affinity for PAK1 compared to the much more complicated, previously established pyridocarbazole complex Λ-FL172. Figure 1 Complicated versus simple design for metal-templated inhibitors of the protein kinase PAK1. Ruthenium complex 1 is based on a simple pyridylphthalimide scaffold, which can be synthesized in just a few steps (Scheme 1). Accordingly, a Suzuki cross-coupling of bromophthalimide 2 with 2-trimethylstannylpyridine afforded the pyridylphthalimide 3 (49%), which was subsequently reacted with the ruthenium complex [Ru(C6H12S3)(MeCN)3](CF3SO3)2 under basic conditions, followed by the addition of NaSCN to afford the racemic complex 1 (38% over two steps). A crystal structure of the N-benzylated derivative of 1 is shown in Figure 2 and demonstrates the formation of a C-Ru bond. This cyclometallation reduces the requirement for metal-coordinating heteroatoms and is thus crucial for the short and efficient synthesis. Figure 2 Structure of the N-benzylated derivative of complex 1 (1Bn). Disordered solvent and a disordered position of S101 are not shown. ORTEP drawing with 50% probability thermal ellipsoids. Selected bond distances (A): C15-Ru1 = 2.033(4), N1-Ru1 = 2.098(3), ... Scheme 1 Synthesis of pyridylphthalimide ruthenium complex 1. The racemic complex 1 displays an IC50 value of 83 ± 20 nM (1 µM ATP) against PAK1, which is slightly more potent than the previously reported Λ-FL172 (IC50 = 130 nM, 1 µM ATP)[4] (Figure 3). Interestingly, the pyridylphthalimide ligand itself is not an inhibitor for PAK1 at all (IC50 > 100 µM), thus demonstrating the importance of the entire coordination sphere for protein kinase binding. Figure 3 IC50 curves with PAK1 of ligand 4, complex 1, and some derivative complexes 5–7. ATP concentration was 1 µM. PAK1 harbors a very atypical, open ATP-binding site which is probably responsible for the difficulties to develop high affinity inhibitors of this kinase.[4,8,10] A cocrystal structure at the resolution of 2.0 A reveals the binding of the (R)-enantiomer of 1 to the ATP binding site of PAK1 (amino acids 249–545 with mutation Lys299Arg)[11] (Figures 4 and ​and5).5). The van der Waals surface representation shown in Figure 4 demonstrates how nicely the ATP-binding site is filled with (R)-1 in a shape complementary fashion with the phthalimide moiety additionally forming two hydrogen bonds with the hinge region (Glu345 and Leu347) and one water-mediated contact to Thr406 (Figure 5). Interestingly, superimposition of this structure with the recently disclosed PAK1/Λ-FL172[4] cocrystal structure demonstrates that even though both compounds are ATP-competitive binders, they significantly differ in there binding modes (Figure 6). While both form the canonical hydrogen bonds between the maleimide moieties of the inhibitor and the hinge region of the kinase, the aromatic heterocycles are rotated towards each other by approximately 30° demonstrating the range that is possible for the directionality of hydrogen bonds. Furthermore, the metals of the two inhibitor scaffolds are 3.0 A apart from each other and point the monodentate ligands into different areas of the active site. Whereas the CO of FL172 is located right below the center of the glycine-rich loop (P-loop), the NCS ligand of 1 points towards the interface of the glycine-rich loop (connecting strands β1 and β2) and the methylene groups of Arg299 of the β-sheet strand β3 and thereby perfectly filling a small hydrophobic pocket as visualized in the van der Waals representation of Figure 4. The importance of the NCS ligand is manifested by the loss of the binding affinity of related complexes, which carry instead a SeCN (IC50 = 0.625 ± 0.06 µM, 7.5-fold weaker inhibitor), NCO (IC50 = 15.7 ± 2 µM, 193-fold weaker inhibitor), or CO (IC50 = 12.7 ± 2 µM, 153-fold weaker inhibitor) ligand (Figure 3). This intriguing example demonstrates how a different position of the metal within the active site, even if it is just shifted by 3.0 A, requires a completely different coordination sphere to fill the protein pocket in a comparable fashion. Figure 4 Cocrystal structure of PAK1 (amino acids 249–545 with mutation Lys299Arg) and (R)-1 at 2.0 A with displayed van der Waals surfaces. Coordinates of the structure have been deposited in the Protein Data Bank (PDB ID: 4DAW). Figure 5 Hydrogen bonding of (R)-1 within the ATP-binding site of PAK1. Figure 6 Relative binding position of (R)-1 and Λ-FL172 (PDB ID: 3FXZ) within the ATP-binding site of PAK1. Superimposed with the PyMOL Molecular Graphics System, Version 1.3, Schrodinger, LLC. In conclusion, the here presented metal-based enzyme inhibitor design together with the analysis of an X-ray cocrystal structure reveals the scope of fitting octahedral metal complexes within an enzyme active site. Despite the shortness of the synthesis (overall only 6 steps) and simplicity of the structure (only two stereoisomers possible), complex 1 displays an IC50 value of 83 nM (1 µM ATP) which is superior to FL172, a complex that is much more tedious to synthesize (>15 steps) and contains a much higher stereochemical complexity (20 possible stereoisomers). To date, the ruthenium phthalimide complex 1 described here belongs to the most potent ATP-competitive inhibitors known for the protein kinase PAK1,[12] demonstrating the advantages of filling large or open pockets with globular octahedral metal complexes.

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.

Development of small-molecule inhibitors of the group I p21-activated kinases, emerging therapeutic targets in cancer

The p21-activated kinases (PAKs), immediate downstream effectors of the small G-proteins of the Rac/cdc42 family, are critical mediators of signaling pathways regulating cellular behaviors and as such, have been implicated in pathological conditions including cancer. Recent studies have validated the requirement for PAKs in promoting tumorigenesis in breast carcinoma and neurofibromatosis. Thus, there has been considerable interest in the development of inhibitors to the PAKs, as biological markers and leads for the development of therapeutics. While initial approaches were based on screening for competitive organic inhibitors, more recent efforts have focused on the identification of allosteric inhibitors, organometallic ATP-competitive inhibitors and the use of PAK1/inhibitor crystal structures for inhibitor optimization. This has led to the identification of highly selective and potent inhibitors, which will serve as a basis for further development of inhibitors for therapeutic applications.

CTCF binding site sequence differences are associated with unique regulatory and functional trends during embryonic stem cell differentiation

CTCF (CCCTC-binding factor) is a highly conserved multifunctional DNA-binding protein with thousands of binding sites genome-wide. Our previous work suggested that differences in CTCF’s binding site sequence may affect the regulation of CTCF recruitment and its function. To investigate this possibility, we characterized changes in genome-wide CTCF binding and gene expression during differentiation of mouse embryonic stem cells. After separating CTCF sites into three classes (LowOc, MedOc and HighOc) based on similarity to the consensus motif, we found that developmentally regulated CTCF binding occurs preferentially at LowOc sites, which have lower similarity to the consensus. By measuring the affinity of CTCF for selected sites, we show that sites lost during differentiation are enriched in motifs associated with weaker CTCF binding in vitro. Specifically, enrichment for T at the 18th position of the CTCF binding site is associated with regulated binding in the LowOc class and can predictably reduce CTCF affinity for binding sites. Finally, by comparing changes in CTCF binding with changes in gene expression during differentiation, we show that LowOc and HighOc sites are associated with distinct regulatory functions. Our results suggest that the regulatory control of CTCF is dependent in part on specific motifs within its binding site.

Structure of the p300 Histone Acetyltransferase Bound to Acetyl-Coenzyme A and Its Analogues.

The p300 and CBP transcriptional coactivator paralogs (p300/CBP) regulate a variety of different cellular pathways, in part, by acetylating histones and more than 70 non-histone protein substrates. Mutation, chromosomal translocation, or other aberrant activities of p300/CBP are linked to many different diseases, including cancer. Because of its pleiotropic biological roles and connection to disease, it is important to understand the mechanism of acetyl transfer by p300/CBP, in part so that inhibitors can be more rationally developed. Toward this goal, a structure of p300 bound to a Lys-CoA bisubstrate HAT inhibitor has been previously elucidated, and the enzyme’s catalytic mechanism has been investigated. Nonetheless, many questions underlying p300/CBP structure and mechanism remain. Here, we report a structural characterization of different reaction states in the p300 activity cycle. We present the structures of p300 in complex with an acetyl-CoA substrate, a CoA product, and an acetonyl-CoA inhibitor. A comparison of these structures with the previously reported p300/Lys-CoA complex demonstrates that the conformation of the enzyme active site depends on the interaction of the enzyme with the cofactor, and is not apparently influenced by protein substrate lysine binding. The p300/CoA crystals also contain two poly(ethylene glycol) moieties bound proximal to the cofactor binding site, implicating the path of protein substrate association. The structure of the p300/acetonyl-CoA complex explains the inhibitory and tight binding properties of the acetonyl-CoA toward p300. Together, these studies provide new insights into the molecular basis of acetylation by p300 and have implications for the rational development of new small molecule p300 inhibitors.

Salicylate, diflunisal and their metabolites inhibit CBP/p300 and exhibit anticancer activity

Salicylate and acetylsalicylic acid are potent and widely used anti-inflammatory drugs. They are thought to exert their therapeutic effects through multiple mechanisms, including the inhibition of cyclo-oxygenases, modulation of NF-κB activity, and direct activation of AMPK. However, the full spectrum of their activities is incompletely understood. Here we show that salicylate specifically inhibits CBP and p300 lysine acetyltransferase activity in vitro by direct competition with acetyl-Coenzyme A at the catalytic site. We used a chemical structure-similarity search to identify another anti-inflammatory drug, diflunisal, that inhibits p300 more potently than salicylate. At concentrations attainable in human plasma after oral administration, both salicylate and diflunisal blocked the acetylation of lysine residues on histone and non-histone proteins in cells. Finally, we found that diflunisal suppressed the growth of p300-dependent leukemia cell lines expressing AML1-ETO fusion protein in vitro and in vivo. These results highlight a novel epigenetic regulatory mechanism of action for salicylate and derivative drugs. DOI: http://dx.doi.org/10.7554/eLife.11156.001

CTCF-Induced Circular DNA Complexes Observed by Atomic Force Microscopy

The CTCF protein has emerged as a key architectural protein involved in genome organization. Although hypothesized to initiate DNA looping, direct evidence of CTCF-induced DNA loop formation is still missing. Several studies have shown that the 11 zinc finger (11 ZF) domain of CTCF is actively involved in DNA binding. We here use atomic force microscopy to examine the effect of the 11 ZF domain comprising residues 266-579 (11 ZF CTCF) and the 3 ZF domain comprising residues 402-494 (6-8 ZF CTCF) of human CTCF on the DNA morphology. Our results show that both domains alter the DNA architecture from the relaxed morphology observed in control DNA samples to compact circular complexes, meshes, and networks, offering important insights into the multivalent character of the 11 ZF CTCF domain. Atomic force microscopy images reveal quasi-circular DNA/CTCF complexes, which are destabilized upon replacing the 11 ZF CTCF by the 6-8 ZF CTCF domain, highlighting the role of the 11 ZF motif in loop formation. Intriguingly, the formation of circular DNA/CTCF complexes is dominated by non-specific binding, whereby contour length and height profiles suggest a single DNA molecule twice wrapped around the protein.