Craig Streu

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.

Catalytic Azide Reduction in Biological Environments

In the quest for the identification of catalytic transformations to be used in chemical biology and medicinal chemistry, we identified iron(III) meso‐tetraarylporphines as efficient catalysts for the reduction of aromatic azides to their amines. The reaction uses thiols as reducing agents and tolerates water, air, and other biological components. A caged fluorophore was employed to demonstrate that the reduction can be performed even in living mammalian cells. However, in vivo experiments in nematodes (Caenorhabditis elegans) and zebrafish (Danio rerio) revealed a limitation to this method: the metabolic reduction of aromatic azides.

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.

Computational design of a β–peptide that targets transmembrane helices

The design of β-peptide foldamers targeting the transmembrane (TM) domains of complex natural membrane proteins has been a formidable challenge. A series of β-peptides was designed to stably insert in TM orientations in phospholipid bilayers. Their secondary structures and orientation in the phospholipid bilayer was characterized using biophysical methods. Computational methods were then devised to design a β-peptide that targeted a TM helix of the integrin α(IIb)β(3). The designed peptide (β-CHAMP) interacts with the isolated target TM domain of the protein and activates the intact integrin in vitro.

Synthesis, Characterization, and Bioactivity of the Photoisomerizable Tubulin Polymerization Inhibitor azo-Combretastatin A4

Combretastatin A4 is a stilbenoid tubulin binding mitotic inhibitor whose conformation greatly influences its potency, making it an excellent candidate for adaptation as a photoactivatable tool. Herein we report a novel synthesis, the facile isomerization with commercial grade equipment, and biological activity of azo-combretastatin A4 in vitro and in human cancer cells. Photoisomerized azo-combretestatin A4 is at least 200-fold more potent in cellular culture, making it a promising phototherapeutic and biomedical research tool.

Writing throughout the Biochemistry Curriculum: Synergistic Inquiry-Based Writing Projects for Biochemistry Students.

This article describes a synergistic two‐semester writing sequence for biochemistry courses. In the first semester, students select a putative protein and are tasked with researching their protein largely through bioinformatics resources. In the second semester, students develop original ideas and present them in the form of a research grant proposal. Both projects involve multiple drafts and peer review. The complementarity of the projects increases student exposure to bioinformatics and literature resources, fosters higher‐order thinking skills, and develops teamwork and communication skills. Student feedback and responses on perception surveys demonstrated that the students viewed both projects as favorable learning experiences.