Structure-Based Screening of Tetrazolylhydrazide Inhibitors versus KDM4 Histone Demethylases

Human histone demethylases are known to play an important role in the development of several tumor types. Consequently, they have emerged as important medical targets for the treatment of human cancer. Herein, structural studies on tetrazolylhydrazide inhibitors as a new scaffold for a certain class of histone demethylases, the JmjC proteins, are reported. A series of compounds are structurally described and their respective binding modes to the KDM4D protein, which serves as a high-resolution model to represent the KDM4 subfamily in crystallographic studies, are examined. Similar to previously reported inhibitors, the compounds described herein are competitors for the natural KDM4 cofactor, 2-oxoglutarate. The tetrazolylhydrazide scaffold fills an important gap in KDM4 inhibition and newly described, detailed interactions of inhibitor moieties pave the way to the development of compounds with high target-binding affinity and increased membrane permeability, at the same time.     

Synthesis and Biological Evaluation of Tripartin,a Putative KDM4 Natural Product Inhibitor,and 1‐Dichloromethylinden‐1‐ol Analogues

The natural product tripartin has been reported to inhibit the N‐methyl‐lysine histone demethylase KDM4A. A synthesis of tripartin starting from 3,5‐dimethoxyphenylacrylic acid was developed, and the enantiomers were separated by chiral HPLC. We observed that both tripartin enantiomers manifested an apparent increase in H3K9me3 levels when dosed in cells, as measured by western blot analysis. Thus, there is no enantiomeric discrimination toward this natural product in terms of its effects on cellular histone methylation status. Interestingly, tripartin did not inhibit isolated KDM4A–E under our assay conditions (IC₅₀>100 μm ). Tripartin analogues with a dichloromethylcarbinol group derived from the indanone scaffold were synthesized and found to be inactive against isolated recombinant KDM4 enzymes and in cell‐based assays. Although the precise cellular mode of action of tripartin is unclear, our evidence suggests that it may affect histone methylation status via a mechanism other than direct inhibition of the KDM4 histone demethylases.     

4‐Biphenylalanine‐ and 3‐Phenyltyrosine‐Derived Hydroxamic Acids as Inhibitors of the JumonjiC‐Domain‐Containing Histone Demethylase KDM4A

Overexpression of the histone lysine demethylase KDM4A, which regulates H3K9 and H3K36 methylation states, has been related to the pathology of several human cancers. We found that a previously reported hydroxamate‐based histone deacetylase (HDAC) inhibitor (SW55) was also able to weakly inhibit this demethylase with an IC₅₀ value of 25.4 μm . Herein we report the synthesis and biochemical evaluations, with two orthogonal in vitro assays, of a series of derivatives of this lead structure. With extensive chemical modifications on the lead structure, also by exploiting the versatility of the radical arylation with aryldiazonium salts, we were able to increase the potency of the derivatives against KDM4A to the low‐micromolar range and, more importantly, to obtain demethylase selectivity with respect to HDACs. Cell‐permeable derivatives clearly showed a demethylase‐inhibition‐dependent antiproliferative effect against HL‐60 human promyelocytic leukemia cells.     

Tetrazolylhydrazides as Selective Fragment‐Like Inhibitors of the JumonjiC‐Domain‐Containing Histone Demethylase KDM4A

The JumonjiC‐domain‐containing histone demethylase 2A (JMJD2A, KDM4A) is a key player in the epigenetic regulation of gene expression. Previous publications have shown that both elevated and lowered enzyme levels are associated with certain types of cancer, and therefore the definite role of KDM4A in oncogenesis remains elusive. To identify a novel molecular starting point with favorable physicochemical properties for the investigation of the physiological role of KDM4A, we screened a number of molecules bearing an iron‐chelating moiety by using two independent assays. In this way, we were able to identify 2‐(1H‐tetrazol‐5‐yl)acetohydrazide as a novel fragment‐like lead structure with low relative molecular mass (M_r=142 Da), low complexity, and an IC₅₀ value of 46.6 μm in a formaldehyde dehydrogenase (FDH)‐coupled assay and 2.4 μm in an antibody‐based assay. Despite its small size, relative selectivity against two other demethylases could be demonstrated for this compound. This is the first example of a tetrazole group as a warhead in JMJD demethylases.     

Peptides Derived from Histone 3 and Modified at Position 18 Inhibit Histone Demethylase KDM6 Enzymes

The KDM6 subfamily of histone lysine demethylases has recently been implicated as a putative target in the treatment of a number of diseases; this makes the availability of potent and selective inhibitors important. Due to high sequence similarity of the catalytic domain of Jumonji C histone demethylases, the development of small‐molecule, family‐specific inhibitors has, however, proven challenging. One approach to achieve the selective inhibition of these enzymes is the use of peptides derived from the substrate, the histone 3 C terminus. Here we used computational methods to optimize such inhibitors of the KDM6 family. Through natural amino acid substitution, it is shown that a K18I variant of a histone H3 derived peptide significantly increases affinity towards the KDM6 enzymes. The crystal structure of KDM6B in complex with a histone 3 derived K18I peptide reveals a tighter fit of the isoleucine side chain, compared with that of the arginine. As a consequence, the peptide R17 residue also has increased hydrophilic interactions. These interactions of the optimized peptide are likely to be responsible for the increased affinity to the KDM6 enzymes.     

Efficient metal‐free aerobic oxidation of aromatic hydrocarbons utilizing aryl‐tetrahalogenated N‐hydroxyphthalimides and 1,4‐diamino‐2,3‐dichloroanthraquinone

BACKGROUND: Hydrocarbon oxidation reactions are central to numerous processes that convert bulk chemicals into useful and higher‐value products. In this investigation, an efficient metal‐free catalytic system for aerobic oxidation of aromatic hydrocarbons was successfully established by synthesizing a series of aryl‐tetrahalogenated N‐hydroxyphthalimides and applying these compounds with 1,4‐diamino‐2,3‐dichloroanthraquinone (DADCAQ). RESULTS: Ethylbenzene was oxidized with 82.3% conversion and 86.9% selectivity to acetophenone catalyzed by the system of TCNHPI/DADCAQ under 0.3 MPa of molecular oxygen at 100 °C for 5 h. Other hydrocarbons were oxidized with high efficiency using this catalytic system. For example, indane can be converted completely to indan‐1‐one with 98.0% selectivity. CONCLUSION: A highly efficient metal‐free catalytic system consisting of TCNHPI and DADCAQ was developed for the oxidation of aromatic hydrocarbons with molecular oxygen. Aryl‐halogen substituents served to significantly increase the activities of the catalytic system. The results in this study can form the basis for the design of an efficient hydrocarbon oxidation process. Copyright © 2008 Society of Chemical Industry     

Stereospecific Effects of Oxygen‐to‐Sulfur Substitution in DNA Phosphate on Ion Pair Dynamics and Protein–DNA Affinity

Oxygen‐to‐sulfur substitutions in DNA phosphate often enhance affinity for DNA‐binding proteins. Our previous studies have suggested that this effect of sulfur substitution of both O_P1 and O_P2 atoms is due to an entropic gain associated with enhanced ion pair dynamics. In this work, we studied stereospecific effects of single sulfur substitution of either the O_P1 or O_P2 atom in DNA phosphate at the Lys57 interaction site of the Antennapedia homeodomain–DNA complex. Using crystallography, we obtained structural information on the R_P and S_P diastereomers of the phosphoromonothioate and their interaction with Lys57. Using fluorescence‐based assays, we found significant affinity enhancement upon sulfur substitution of the O_P2 atom. Using NMR spectroscopy, we found significant mobilization of the Lys57 side‐chain NH₃⁺ group upon sulfur substitution of the O_P2 atom. These data provide further mechanistic insights into the affinity enhancement by oxygen‐to‐sulfur substitution in DNA phosphate.     

Interleukin‐4‐Clicked Surfaces Drive M2 Macrophage Polarization

Driving macrophage (M?) polarization into the M2 phenotype provides potential against inflammatory diseases. Interleukin‐4 (IL‐4) promotes polarization into the M2‐M? phenotype, but its systemic use is constrained by dose‐limiting toxicity. Consequently, we developed IL‐4‐decorated surfaces aiming at sustained and localized activity. IL‐4 muteins were generated by genetic code expansion; Lys42 was replaced by unnatural amino acids (uAAs). Both muteins showed cell‐stimulation ability and binding affinity to IL4Rα similar to those of wt‐IL‐4. Copper‐catalyzed (CuAAC) and copper‐free strain‐promoted (SPAAC) 1,3‐dipolar azide–alkyne cycloadditions were used to site‐selectively anchor IL‐4 to agarose surfaces. These surfaces had sustained IL‐4 activity, as demonstrated by TF‐1 cell proliferation and M2, but not M1, polarization of M‐CSF‐generated human M?. The approach provides a blueprint for the engineering of cytokine‐activated surfaces profiled for sustained and spatially controlled activity.     

Peptide Architecture: Adding an α‐Helix to the PYY Lysine Side Chain Provides Nanomolar Binding and Body‐Weight‐Lowering Effects

The gut hormone PYY3‐36 influences food intake and body weight via interaction with hypothalamic presynaptic Y2 receptors (Y2R). Novel Y2R‐selective analogues of PYY3‐36 are therefore potential drug candidates for the treatment of obesity. It has been hypothesized that PYY3‐36 and possibly also the related PP‐fold peptides, NPY and PP, bind to the membrane via their amphipathic α‐helix prior to receptor interaction. The PYY3‐36 amphipathic α‐helix causes the peptide to associate with the membrane, making it essential for Y receptor potency as it potentially guides the C‐terminal pentapeptide into the correct conformation for receptor activation. Based on this hypothesis, the importance of the amphipathic nature of PYY3‐36, as well as the ability of amphipathic α‐helices to interact in solution to form di‐ and tetramers, we redesigned the peptide architecture by addition of an amphipathic α‐helix via the Lys 4 side chain of PYY3‐36. Two different amphipathic sequences were introduced; first, PYY17‐31, the native α‐helix of PYY, and secondly, its retro counterpart, PYY31‐17, which is also predicted to form an α‐helix. Moreover, several different turn motifs between the branching point and the additional α‐helix were tested. Several novel peptides with nanomolar Y2R binding affinities, as well as increased Y receptor selectivity, were identified. CD experiments showed the modifications to be well accepted, and an increase in mean ellipticity (ME) signifying an increased degree of α‐helicity was observed. Receptor binding experiments indicated that the direction of the additional α‐helix is less important, in contrast to the turn motifs, which greatly affect the Y1R binding and thus determine the Y1R activity. Conversely, the structure–activity relationships from in vivo data showed that the peptide containing the retro‐sequence was inactive, even though the binding data demonstrated high affinity and selectivity. This demonstrates that radical redesign of peptide architecture can provide nanomolar binding with improved subtype selectivity and with in vivo efficacy.     

Self‐Generating High‐Temperature Oxidation‐Resistant Glass‐Ceramic Coatings for C–C Composites Using UHTCs

Carbon–carbon (C–C) composites are ideal for use as aerospace vehicle structural materials; however, they lack high‐temperature oxidation resistance requiring environmental barrier coatings for application. Ultra high‐temperature ceramics (UHTCs) form oxides that inhibit oxygen diffusion at high temperature are candidate thermal protection system materials at temperatures >1600°C. Oxidation protection for C–C composites can be achieved by duplicating the self‐generating oxide chemistry of bulk UHTCs formed by a “composite effect” upon oxidation of ZrB₂–SiC composite fillers. Dynamic Nonequilibrium Thermogravimetric Analysis (DNE‐TGA) is used to evaluate oxidation in situ mass changes, isothermally at 1600°C. Pure SiC‐based fillers are ineffective at protecting C–C from oxidation, whereas ZrB₂–SiC filled C–C composites retain up to 90% initial mass. B₂O₃ in SiO₂ scale reduces initial viscosity of self‐generating coating, allowing oxide layer to spread across C–C surface, forming a protective oxide layer. Formation of a ZrO₂–SiO₂ glass‐ceramic coating on C–C composite is believed to be responsible for enhanced oxidation protection. The glass‐ceramic coating compares to bulk monolithic ZrB₂–SiC ceramic oxide scale formed during DNE‐TGA where a comparable glass‐ceramic chemistry and surface layer forms, limiting oxygen diffusion.     

Highly efficient and organic nitrogen‐containing cation‐promoted aerobic oxidation of alkylaromatics in the presence of N‐hydroxyphthalimide

BACKGROUD: Direct oxidation of alkylaromatics to the corresponding aromatic ketone is an important process in the manufacture of perfumes, pharmaceuticals, flavors, dyes and agrochemicals. For example, tetralin is oxidized to produce 1‐tetralinone, which is a key intermediate in the commercial production of 1‐naphthol, 2‐hydroxy‐1‐tetralone, aureolic acid antibiotics and other pharmaceuticals. RESULTS: In this investigation, it was found that alkylaromatics could be efficiently and selectively oxidized to the corresponding aromatic ketones when methyl violet was employed as a promotor in the presence of N‐hydroxyphthalimide (NHPI); tetralin was oxidized with 89% conversion and 76% selectivity to 1‐tetralinone under 0.3 MPa of O₂ at 75 °C for 2.5 h. The effects of temperature, oxygen pressure, reaction time and additive inclusion were studied in detail. A possible reaction mechanism for tetralin oxidation has been proposed. CONCLUSION: It was demonstrated that methyl violet could efficiently promote the aerobic oxidation of alkylaromatics in the presence of NHPI under mild conditions. Further investigations indicated that the nitrogen cation had a crucial promotion effect in the oxidation process. Copyright © 2009 Society of Chemical Industry     

Sodium azide and metal chelator effects on 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH) radical scavenging compounds from methylene blue photosensitized lard

Thermal oxidation of edible oils can generate 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH) radical scavenging compounds from oxidized lipids (RSOLs). However, effects of photosensitization on the formation of RSOLs have not been reported yet. Methylene blue (MB) photosensitization and involvement of singlet oxygen and transition metals on the RSOL formations were determined in stripped lard oils. RSOLs were formed in lard containing MB and visible light irradiation only. Addition of sodium azide decreased RSOLs with concentration dependent manner, which implies singlet oxygen was involved on the RSOL formation. Ethylenediammetetraacetic acid (EDTA), a well known metal chelator, accelerated the formation of RSOLs through protecting the decomposition of MB photosensitizer. Results from p‐anisidine values showed that RSOLs from photosensitization may not be formed from the same pathways compared to thermal oxidation. Practical application: Understanding mechanisms of lipid oxidation can help extend the shelf‐life of foods. Photosensitization plays important roles in accelerating the rates of lipid oxidation. The results of this study showed that foods containing photosensitizers can generate radical scavenging compounds from oxidized lipids (RSOLs) under visible light irradiation and singlet oxygen is involved in the formations of these compounds. However, these compounds may not share the same pathways with thermally oxidized lipids. Metal chelating agents accelerated the rates of lipid oxidation and formation of RSOLs which implies that metal chelators can act as prooxidant. Careful considerations are necessary on the addition of metal chelators because non‐polar photosensitizers can act a prooxidant.     

Enzymatic Redox Cascade for One‐Pot Synthesis of Uridine 5′‐Diphosphate Xylose from Uridine 5′‐Diphosphate Glucose

Synthetic ways towards uridine 5′‐diphosphate (UDP)‐xylose are scarce and not well established, although this compound plays an important role in the glycobiology of various organisms and cell types. We show here how UDP‐glucose 6‐dehydrogenase (hUGDH) and UDP‐xylose synthase 1 (hUXS) from Homo sapiens can be used for the efficient production of pure UDP‐α‐xylose from UDP‐glucose. In a mimic of the natural biosynthetic route, UDP‐glucose is converted to UDP‐glucuronic acid by hUGDH, followed by subsequent formation of UDP‐xylose by hUXS. The nicotinamide adenine dinucleotide (NAD⁺) required in the hUGDH reaction is continuously regenerated in a three‐step chemo‐enzymatic cascade. In the first step, reduced NAD⁺ (NADH) is recycled by xylose reductase from Candida tenuis via reduction of 9,10‐phenanthrenequinone (PQ). Radical chemical re‐oxidation of this mediator in the second step reduces molecular oxygen to hydrogen peroxide (H₂O₂) that is cleaved by bovine liver catalase in the last step. A comprehensive analysis of the coupled chemo‐enzymatic reactions revealed pronounced inhibition of hUGDH by NADH and UDP‐xylose as well as an adequate oxygen supply for PQ re‐oxidation as major bottlenecks of effective performance of the overall multi‐step reaction system. Net oxidation of UDP‐glucose to UDP‐xylose by hydrogen peroxide (H₂O₂) could thus be achieved when using an in situ oxygen supply through periodic external feed of H₂O₂ during the reaction. Engineering of the interrelated reaction parameters finally enabled production of 19.5 mM (10.5 g L ⁻¹) UDP‐α‐xylose. After two‐step chromatographic purification the compound was obtained in high purity (>98%) and good overall yield (46%). The results provide a strong case for application of multi‐step redox cascades in the synthesis of nucleotide sugar products.

     

Facile Fluorescence‐Based Detection of PAD4‐Mediated Citrullination

The post‐translational modifications of histone proteins are highly diverse and dynamic processes. It is becoming increasingly evident that modifying histone proteins can have a direct influence on both cellular homeostasis and disease states. Protein arginine deiminase 4 (PAD4) is an enzyme that converts peptidyl‐arginine to citrulline. The overexpression of PAD4 has been found in numerous types of human cancer and autoimmune diseases. We report a new, facile, fluorescence‐based assay for the detection of PAD4 activity that exploits the substrate specificity of trypsin to monitor the citrullination reaction carried out by PAD4 based on the fact that, upon citrullination, the positively charged arginine side chain is converted to the neutral citrulline. We show that the assay can be performed rapidly with readily available reagents and that it responds accordingly to a known PAD4 inhibitor.     

Comparative photo‐oxidation under natural and accelerated conditions of polypropylene nanocomposites produced by extrusion and injection molding

The photo‐oxidation behavior under natural and accelerated conditions of polypropylene/layered silicate nanocomposite is studied in this article. The nanocomposites are prepared via simple melt mixing (extrusion and injection molding). The structure obtained is very dependent on the preparation mode and the modified clay used; mostly, exfoliation structure is produced. The nanocomposites start their photo‐degradation earlier than the control samples polypropylene and polypropylene‐graft‐maleic anhydride with a higher oxidation rate for specimen produced by injection molding. This is explained by the presence of organiphilic‐modified montmorillonite layers that trap the oxygen, increasing the oxygen pressure in the bulk and leading to a decrease of the induction period. Contrary to the control samples that display auto acceleration in their oxidation kinetics, the nanocomposites show a slight tendency to a plateau indicating a slowing down of the photo‐oxidation process. This is ascribed to oxygen starvation that occurs in the nanocomposite. The acceleration factor is found to be higher for the nanocomposite comparatively of the control samples. With the aid of SF₄ and NO treatments, the mechanism of photo degradation was found to be similar in PPgMA and its nanocomposites. © 2007 Wiley Periodicals, Inc. J Appl Polym Sci, 2007     

Alteration of the Diastereoselectivity of 3‐Methylaspartate Ammonia Lyase by Using Structure‐Based Mutagenesis

3‐Methylaspartate ammonia‐lyase (MAL) catalyzes the reversible amination of mesaconate to give both (2S,3S)‐3‐methylaspartic acid and (2S,3R)‐3‐methylaspartic acid as products. The deamination mechanism of MAL is likely to involve general base catalysis, in which a catalytic base abstracts the C3 proton of the respective stereoisomer to generate an enolate anion intermediate that is stabilized by coordination to the essential active‐site Mg^II ion. The crystal structure of MAL in complex with (2S,3S)‐3‐methylaspartic acid suggests that Lys331 is the only candidate in the vicinity that can function as a general base catalyst. The structure of the complex further suggests that two other residues, His194 and Gln329, are responsible for binding the C4 carboxylate group of (2S,3S)‐3‐methylaspartic acid, and hence are likely candidates to assist the Mg^II ion in stabilizing the enolate anion intermediate. In this study, the importance of Lys331, His194, and Gln329 for the activity and stereoselectivity of MAL was investigated by site‐directed mutagenesis. His194 and Gln329 were replaced with either an alanine or arginine, whereas Lys331 was mutated to a glycine, alanine, glutamine, arginine, or histidine. The properties of the mutant proteins were investigated by circular dichroism (CD) spectroscopy, kinetic analysis, and ¹H NMR spectroscopy. The CD spectra of all mutants were comparable to that of wild‐type MAL, and this indicates that these mutations did not result in any major conformational changes. Kinetic studies demonstrated that the mutations have a profound effect on the values of k_cat and k_cat/K_M; this implicates Lys331, His194 and Gln329 as mechanistically important. The ¹H NMR spectra of the amination and deamination reactions catalyzed by the mutant enzymes K331A, H194A, and Q329A showed that these mutants have strongly enhanced diastereoselectivities. In the amination direction, they catalyze the conversion of mesaconate to yield only (2S,3S)‐3‐methylaspartic acid, with no detectable formation of (2S,3R)‐3‐methylaspartic acid. The results are discussed in terms of a mechanism in which Lys331, His194, and Gln329 are involved in positioning the substrate and in formation and stabilization of the enolate anion intermediate.     

Treatment of Fenton‐refractory olive oil mill wastes by electrochemical oxidation with boron‐doped diamond anodes

In this work, the electrochemical oxidation of an actual industrial waste with conductive diamond anodes has been studied. The wastewater is the effluent of a wastewater treatment plant consisting of a Fenton reactor followed by a settler and a sand filter, in which the wastes generated in an olive oil mill are treated. These wastes contain a residual chemical oxygen demand of nearly 700 mg dm^?3 which cannot be further oxidized with the Fenton process. The electrolyses were carried out under galvanostatic conditions, using a bench‐scale plant equipped with a single‐compartment electrochemical flow cell. Boron‐doped diamond (BDD) and stainless steel (AISI 304) were use as anode and cathode of the cell, respectively. The complete mineralization of the waste was obtained with high current efficiencies limited only by mass transport processes. This confirms that besides the hydroxyl radical‐mediated oxidation that occurs in the Fenton process, the electrochemical oxidation with conductive diamond electrodes combines other important oxidation processes such as direct electro‐oxidation on the BDD surface and oxidation mediated by other electrochemically formed compounds generated in this electrode. Copyright © 2006 Society of Chemical Industry     

Enzymatic Methylation and Structure–Activity‐Relationship Studies on Polycarcin V,a Gilvocarcin‐Type Antitumor Agent

Polycarcin V, a polyketide natural product of Streptomyces polyformus, was chosen to study structure–activity relationships of the gilvocarcin group of antitumor antibiotics due to a similar chemical structure and comparable bioactivity with gilvocarcin V, the principle compound of this group, and the feasibility of enzymatic modifications of its sugar moiety by auxiliary O‐methyltransferases. Such enzymes were used to modify the interaction of the drug with histone H3, the biological target that interacts with the sugar moiety. Cytotoxicity assays revealed that a free 2′‐OH group of the sugar moiety is essential to maintain the bioactivity of polycarcin V, apparently an important hydrogen bond donor for the interaction with histone H3, and converting 3′‐OH into an OCH₃ group improved the bioactivity. Bis‐methylated polycarcin derivatives revealed weaker activity than the parent compound, indicating that at least two hydrogen bond donors in the sugar are necessary for optimal binding.