Cysteine-179 of IkappaB kinase beta plays a critical role in enzyme activation by promoting phosphorylation of activation loop serines

IkappaB kinase beta (IKKbeta) subunit of IKK complex is essential for the activation of NF-kappaB in response to various proinflammatory signals. Cys-179 in the activation loop of IKKbeta is known to be the target site for IKK inhibitors such as cyclopentenone prostaglandins, arsenite, and antirheumatic gold compounds. Here we show that a mutant IKKbeta in which Cys-179 is substituted with alanine had decreased activity when it was expressed in HEK-293 cells, and TNF stimulation did not restore the activity. Phosphorylation of activation loop serines (Ser-177 and Ser-181) which is required for IKKbeta activation was reduced in the IKKbeta (C179A) mutant. The activity of IKKbeta (C179A) was partially recovered when its phosphorylation was enforced by coexpression with mitogen-activated protein kinase kinase kinases (MAPKKK) such as NF-kappaB inducing kinase (NIK) and MAPK/extracellular signal-regulated kinase kinase kinase 1(MEKK1) or when the serine residues were replaced with phospho-mimetic glutamate. The IKKbeta (C179A) mutant was normal in dimer formation, while its activity abnormally responded to the change in the concentration of substrate ATP in reaction mixture. Our results suggest that Cys-179 of IKKbeta plays a critical role in enzyme activation by promoting phosphorylation of activation-loop serines and interaction with ATP.     

Dual effect of oxidative stress on NF-kappakB activation in HeLa cells

Reactive oxygen species (ROS) has been implicated as an inducer of NF-kappaB activity in numbers of cell types where exposure of cells to ROS such as H(2)O(2) leads to NF-kappaB activation. In contrast, exposure to oxidative stress in certain cell types induced reduction of tumor necrosis factor (TNF)- induced NF-kappaB activation. And various thiol-modifying agents including gold compounds and cyclopentenone prostaglandins inhibit NF-kappaB activation by blocking IkappaB kinase (IKK). To understand such conflicting effect of oxidative stress on NF- kappakB activation, HeLa cells were incubated with H(2)O(2) or diamide and TNF-induced expression of NF-kappaB reporter gene was measured. NF-kappaB activation was significantly blocked by these oxidizing agents, and the inhibition was accompanied with reduced nuclear NF-kappaB and inappropriate cytosolic IkappaB degradation. H(2)O(2) and diamide also inhibited IKK activation in HeLa and RAW 264.7 cells stimulated with TNF and lipopolysaccharide, respectively, and directly blocked IKK activity in vitro. In cells treated with H(2)O(2) alone, nuclear NF-kappaB was induced after 2 h without detectable degradation of cytosolic IkappaBalphaa or activation of IKK. Our results suggest that ROS has a dual effect on NF-kappaB activation in the same HeLa cells: it inhibits acute IKK-mediated NF-kappakB activation induced by inflammatory signals, while longer-term exposure to ROS induces NF-kappaB activity through an IKK-independent pathway.     

The anti-inflammatory natural product parthenolide from the medicinal herb Feverfew directly binds to and inhibits IkappaB kinase

BACKGROUND: Biologically active natural products continue to be useful in the exploration and control of intracellular signaling processes. For example, the sesquiterpene lactone parthenolide from the anti-inflammatory medicinal herb Feverfew (Tanacetum parthenium) appears to inhibit the pro-inflammatory signaling pathway. Parthenolide's direct molecular target, however, remains unknown. We set out to identify the molecular mechanisms of parthenolide's anti-inflammatory activity. RESULTS: A parthenolide affinity reagent was synthesized and shown to bind directly to and inhibit IkappaB kinase beta (IKKbeta), the kinase subunit known to play a critical role in cytokine-mediated signaling. Mutation of cysteine 179 in the activation loop of IKKbeta abolished sensitivity towards parthenolide. Moreover, we showed that parthenolide's in vitro and in vivo anti-inflammatory activity is mediated through the alpha-methylene gamma-lactone moiety shared by other sesquiterpene lactones. CONCLUSIONS: In recent years, the multi-subunit IKK complex has been shown to be responsible for cytokine-mediated stimulation of genes involved in inflammation and as such represents an attractive target for pharmaceutical intervention. Our finding that parthenolide targets this kinase complex provides a possible molecular basis for the anti-inflammatory properties of parthenolide. In addition, these results may be useful in the development of additional anti-inflammatory agents.     

Synthesis and evaluation of novel aza-caged Garcinia xanthones

Inspired by the therapeutic potential of the simplified caged xanthones, we have developed a chemical strategy for synthesizing novel aza-caged Garcinia analogues through a regioselective Claisen/Diels-Alder cascade reaction. The origin of regioselectivity has been explained using the DFT method. We have further evaluated the cell proliferation and IKKβ inhibitory activities of these compounds and studied their binding mode with IKKβ by molecular docking. The results suggested that the aza-caged scaffold provides a suitable modification site and the introduction of a hydrophobic moiety leads to improvement in the cytotoxicity and IKKβ inhibitory activity. The aza-caged compound 6c exhibited an IC(50) value of 2.68, 2.10, 8.02 μM against the HepG2, A549 cells and IKKβ, respectively. Mechanism studies with 6c showed that the aza-caged compounds induce apoptosis and cell cycle S phase arrest in A549 cells.     

Molecular modeling and mutagenesis reveals a tetradentate binding site for Zn2+ in GABA(A) alphabeta receptors and provides a structural basis for the modulating effect of the gamma subunit

Gamma-aminobutyric acid type A receptors (GABA(A)-R) containing alpha1beta2gamma2 subunits are weakly inhibited by Zn2+, whereas receptors containing only the alpha1beta2 subunits are strongly inhibited. We built homology models of the ion pores of alpha1beta2 and alpha1beta2gamma2 GABA(A)-R using coordinates of the nicotinic acetylcholine receptor as a template. Threading the GABA(A)-R beta2 sequence onto this template placed the 17' histidine and the 20' glutamate residues at adjacent locations in the mouth of the pore, such that a nearly ideal tetradentate site for Zn2+ was formed from two histidine and two glutamate residues between adjacent beta subunits in the alpha1beta2 GABA(A)-R. Following optimization with CHARMM, the distance between the alpha-carbons of the adjacent histidine residues was approximately 9.2 A, close to the ideal distance for a Zn2+ binding site. Loss of inhibition by Zn2+ in alpha1beta2gamma2 GABA(A)-R can be explained by the geometry of these residues in the arrangement alpha1beta2gamma2alpha1beta2, in which the nearest C-alpha-C-alpha distance between the histidine residues is 15.5 A, too far apart for an energetically optimal Zn2+ binding site. We then mutated the gamma subunit at the 17' and/or 20' positions. Zn2+ inhibition was not restored in alpha1beta2gamma2 (I282H) receptors. A novel finding is that the modeling shows the native 20' lysine in gamma2 can compete with Zn2+ for binding to the inserted 17' histidine. Sensitivity to Zn2+ was restored in the double mutant receptor, alpha1beta2gamma2 (I282H; K285E), in which the competition with lysine was removed and a more favorable Zn2+ binding site was formed.     

Formation of a cationic gold(I) complex and disulfide by oxidation of the antiarthritic gold drug auranofin

The mechanism of action of auranofin, an antiarthritic gold(I) drug, is unknown, but several studies suggest that oxidation may be important for its biochemical effect. Bulk electrolysis studies on auranofin [(Et(3)P)Au(TATG); TATG = 2,3,4,6-tetraacetyl-1-thio-d-glucopyranosato] at +1.2 and +1.6 V versus Ag/AgCl in 0.1 M Bu(4)NBF(4)/CH(2)Cl(2) results in n values of 0.5 and >2 electrons, respectively. Oxidation of auranofin with the mild oxidant, Cp(2)Fe(+), results in formation of disulfide and a digold(I) cation with a bridging thiolate ligand, [(Et(3)PAu)(2)(mu-TATG)](+) (1). The X-ray structure of the PMe(3) analogue, [(Me(3)PAu)(2)(mu-TATG)](NO(3)) (2), is reported. Compound 2 forms a tetranuclear cluster containing an almost perfect square of four gold atoms with Au.Au distances averaging 3.14 A. The complex crystallizes in the tetragonal space group P4(2)2(1)2 with cell constants a = 26.1758(6) A, b = 26.1758(6) A, c = 9.7781(3) A, alpha = beta = gamma = 90 degrees, V = 6699.7(3) A(3), Z = 4, R1 = 0.0644, and wR2 = 0.1152. A mechanism for oxidation of auranofin and possible biological implications are discussed.     

Synthesis and structure activity relationship studies of benzothieno[3,2-b]furan derivatives as a novel class of IKKbeta inhibitors

As a novel class of IKKbeta inhibitors, a series of tricyclic furan derivatives was designed and synthesized based on the structure of known thiophene IKKbeta inhibitors. Among the various fused furan derivatives synthesized, a benzothieno[3,2-b]furan derivative 13a displayed potent inhibitory activity towards IKKbeta in enzymatic and cellular assays. The potent inhibitory activity originates from an intramolecular non-bonded S...O interaction which was confirmed by the X-ray structure of JNK3 with 16k. The introduction of further substituents on the core structure led to the discovery of the 6-alkoxy derivatives, which possessed a comparable IKKbeta inhibitory activity to 13a and an improved metabolic stability. Among these, appropriately lipophilic compounds 16a, h, i, and 13g (log D>2) were found to possess good oral bioavailability.     

Glyco‐pseudopolyrotaxanes: Carbohydrate Wheels Threaded on a Polymer String and Their Inhibition of Bacterial Adhesion

We report glyco‐pseudopolyrotaxanes composed of cucurbit[6]uril‐based mannose wheels (ManCB[6]) threaded on polyviologen (PV), which not only effectively induce bacterial aggregation, but also exhibit high inhibitory activity against bacterial binding to host cells. Three glyco‐pseudopolyrotaxanes ( 1 – 3 ), which have 10, 5, and 3 ManCB[6] wheels, respectively, on a PV string, were prepared and characterized. Bacterial aggregation assays and hemagglutination inhibition assays illustrated the specific and multivalent interaction between the glyco‐pseudopolyrotaxanes and E. coli ORN178. Compound 3 was especially effective at inducing bacterial aggregation and showed 300 times higher inhibitory potency than monomeric methyl‐α‐mannoside (Me‐αMan) for ORN178‐induced hemagglutination. Furthermore, we demonstrated their inhibitory activities for the adhesion of ORN178 bacteria to urinary epithelial cells as a model of urinary tract infection. Our findings suggest that these supramolecular carbohydrate clusters are potentially useful in antiadhesion therapy.     

Unveiling the Potential of Innovative Gold(I) and Silver(I) Selenourea Complexes as Anticancer Agents Targeting TrxR and Cellular Redox Homeostasis

A series of NHC-based selenourea Ag(I) and Au(I) complexes were evaluated for their anticancer potential in vitro, on 2D and 3D human cancer cell systems. All NHC-based selenourea complexes possess an outstanding cytotoxic potency, which was comparable or even better than that of the reference metallodrug auranofin, and were also able to overcome both platinum-based and multi-drug resistances. Intriguingly, their cytotoxic potency did not correlate with solution stability, partition coefficient or cellular uptake. On the other hand, mechanistic studies in cancer cells revealed their ability to strongly and selectively inhibit the redox-regulating enzyme Thioredoxin Reductase (TrxR), being even more effective than auranofin, a well-known TrxR inhibitor, without affecting other redox enzymes such as Glutathione Reductase (GR). The inhibition of TrxR in H157 human cancer cells caused, in turn, the disruption of cellular thiol-redox homeostasis and of mitochondria pathophysiology, ultimately leading to cancer cell death through apoptosis.     

Extended peptide-based inhibitors efficiently target the proteasome and reveal overlapping specificities of the catalytic beta-subunits

BACKGROUND: The 26S proteasome is responsible for most cytosolic proteolysis, and is an important protease in major histocompatibility complex class I-mediated antigen presentation. Constitutively expressed proteasomes from mammalian sources possess three distinct catalytically active species, beta1, beta2 and beta5, which are replaced in the gamma-interferon-inducible immunoproteasome by a different set of catalytic subunits, beta1i, beta2i and beta5i, respectively. Based on preferred cleavage of short fluorogenic peptide substrates, activities of the proteasome have been assigned to individual subunits and classified as 'chymotryptic-like' (beta5), 'tryptic-like' (beta2) and 'peptidyl-glutamyl peptide hydrolyzing' (beta1). Studies with protein substrates indicate a far more complicated, less strict cleavage preference. We reasoned that inhibitors of extended size would give insight into the extent of overlapping substrate specificity of the individual activities and subunits. RESULTS: A new class of proteasome inhibitors, considerably extended in comparison with the commonly used fluorescent substrates and peptide-based inhibitors, has been prepared. Application of the safety catch resin allowed the generation of the target compounds using a solid phase protocol. Evaluation of the new compounds revealed a set of highly potent proteasome inhibitors that target all individual active subunits with comparable affinity, unlike the other inhibitors described to date. Modification of the most active compound, adamantane-acetyl-(6-aminohexanoyl)(3)-(leucinyl)(3)-vinyl-(methyl)-sulfone (AdaAhx(3)L(3)VS), itself capable of proteasome inhibition in living cells, afforded a new set of radio- and affinity labels. CONCLUSIONS: N-terminal extension of peptide vinyl sulfones has a profound influence on both their efficiency and selectivity as proteasome inhibitors. Such extensions greatly enhance inhibition and largely obliterate selectivity towards the individual catalytic activities. We conclude that for the interaction with larger substrates, there appears to be less discrimination of different substrate sequences for the catalytic activities than is normally assumed based on the use of small peptide-based substrates and inhibitors. The compounds described here are readily accessible synthetically, and are more potent inhibitors in living cells than their shorter peptide vinyl sulfone counterparts.     

In vitro inhibitory properties of ferrocene-substituted chalcones and aurones on bacterial and human cell cultures

Two series of ten chalcones and ten aurones, where ferrocene replaces the C ring and with diverse substituents on the A ring were synthesized. The compounds were tested against two antibiotic-sensitive bacterial strains, E. coli ATCC 25922 and S. aureus ATCC 25923, and two antibiotic-resistant strains, S. aureus SA-1199B and S. epidermidis IPF896. The unsubstituted compound and those with methoxy substitution showed an inhibitory effect on all bacterial strains at minimum inhibitory concentrations ranging between 2 and 32 mg L(-1). For four of these compounds, the effect was bactericidal, as opposed to bacteriostatic. The corresponding organic aurones did not show growth inhibition, underscoring the role of the ferrocene group. The methoxy-substituted aurones and the unsubstituted aurone also showed low micromolar (IC(50)) activity against MRC-5 non-tumoral lung cells and MDA-MB-231 breast cancer cells, suggesting non-specific toxicity.     

Kinetics and Mechanism of the Reaction between Serum Albumin and Auranofin (and Its Isopropyl Analogue) in Vitro

The first detailed kinetic analysis and mechanistic interpretation of the reactions between serum albumin and the second-generation gold drug Auranofin [Et(3)PAuSATg = (triethylphosphine)(2,3,4,6-tetra-O-acetyl-1-beta-D-glucopyranosato-S-) gold(I)] and its triisopropylphosphine analogue, iPr(3)PAuSATg, in vitro are reported. The reactions were investigated using Penefsky spun columns and NMR saturation transfer methods. Under the Penefsky chromatography conditions with 0.4-0.6 mM albumin and a wide range of Et(3)PAuSATg concentrations, the reaction is biphasic. The fast phase is apparently first order in albumin with a rate constant [k(1) = 3.4 +/- 0.3 x 10(-)(2) s(-)(1)] that decreases slightly in magnitude and becomes intermediate in order at low gold concentrations, [Et(3)PAuSATg] < [AlbSH]; it accounts for approximately 95% of the Au(I) that binds. A minor, slower step [k(2) = 2.3 +/- 0.3 x 10(-)(3) s(-)(1)), which accounts for only 5% of the reaction, is also first order with respect to albumin, and zero order with respect to auranofin. For iPr(3)PAuSATg, only the first step was observed, k(1) = (1.4 +/- 0.1) x 10(-)(2) s(-)(1), and is first order in albumin and independent of the iPr(3)PAuSATg concentration. (31)P-NMR saturation transfer experiments utilizing iPr(3)PAuSATg, under equilibrium conditions, yielded second-order rate constants for both the forward (1.2 x 10(2) M(-)(1) s(-)(1)) and the reverse (3.9 x 10(1) M(-)(1) s(-)(1)) directions. A multistep mechanism involving a conformationally altered albumin species was developed. Albumin domain IA opens with concomitant Cys-34 rearrangement, allowing facile gold binding and exchange, and then closes. In conjunction with the steady-state approximation, this mechanism accounts for the different reaction orders observed under the two set of conditions. The rate-determining conformational change of albumin governs the reaction as monitored by the Penefsky columns. Rapid second order exchange of R(3)PAuSATg at the exposed Cys-34 residue is observed under the NMR conditions. The mechanism predicts that under physiological conditions where [Et(3)PAuSATg] is 10-25 &mgr;M, the reaction will be second order and rapid with a rate constant of 8 +/- 2 x 10(2) M(-)(1) s(-)(1). The Penefsky spun columns revealed a previously unreported and novel binding mechanism, association of auranofin in the pocket of albumin-disulfide species, which was confirmed by Hummel-Dreyer gel chromatographic techniques under equilibrium conditions. This albumin-auranofin complex (AlbSSR-Et(3)PAuSATg) is weakly bound and readily dissociates during conventional gel exclusion chromatography.     

1,2,4‐Triazole‐based N‐heterocyclic carbene complexes of gold(I): synthesis,characterization and biological activity

Two gold(I) complexes of the (NHC)AuX type bearing a triazole‐based N‐heterocyclic carbene (NHC) ligand (1‐tert‐butyl‐4‐(4‐methylphenyl)‐3‐phenyl‐1H‐1,2,4‐triazol‐4‐ium‐5‐ylidene) and various halide ligands (X = Br, I) were synthesized and characterized in solution using NMR spectroscopy as well as in the solid state using X‐ray diffraction techniques. The cytotoxic properties of both compounds and the precursor, (NHC)AuCl, were screened against a panel of human tumour cell lines including liver cancer (HepG2), cervical cancer (HeLa S3) and leukaemia (CCRF‐CEM, HL‐60) and compared to cisplatin and auranofin. It was found that the activities of the chloro and bromo derivatives were generally superior to that of cisplatin and slightly less effective compared to auranofin, except for HepG2 cells where auranofin was not as effective. In addition, the ability to induce membrane phosphatidyl serine externalization as a hallmark of apoptosis in CCRF‐CEM leukaemic cells was investigated. Copyright © 2016 John Wiley & Sons, Ltd.     

Coptidis rhizoma extract protects against cytokine-induced death of pancreatic beta-cells through suppression of NF-kappaB activation

We demonstrated previously that Coptidis rhizoma extract (CRE) prevented S-nitroso-N-acetylpenicillamine-induced apoptotic cell death via the inhibition of mitochondrial membrane potential disruption and cytochrome c release in RINm5F (RIN) rat insulinoma cells. In this study, the preventive effects of CRE against cytokine-induced beta-cell death was assessed. Cytokines generated by immune cells infiltrating pancreatic islets are crucial mediators of beta-cell destruction in insulin-dependent diabetes mellitus. The treatment of RIN cells with IL-1beta and IFN-gamma resulted in a reduction of cell viability. CRE completely protected IL-1beta and IFN-gamma-mediated cell death in a concentration-dependent manner. Incubation with CRE induced a significant suppression of IL-1beta and IFN-gamma-induced nitric oxide (NO) production, a finding which correlated well with reduced levels of the iNOS mRNA and protein. The molecular mechanism by which CRE inhibited iNOS gene expression appeared to involve the inhibition of NF-kappaB activation. The IL-1beta and IFN-gamma-stimulated RIN cells showed increases in NF-kappaB binding activity and p65 subunit levels in nucleus, and IkappaB alpha degradation in cytosol compared to unstimulated cells. Furthermore, the protective effects of CRE were verified via the observation of reduced NO generation and iNOS expression, and normal insulin-secretion responses to glucose in IL-1beta and IFN-gamma-treated islets.     

Synthesis and Characterization of Pyrazoleanthrone Derivatives as Aurora A Kinase Inhibitors

Aurora A is a cell cycle kinase linked to cancer. For the purpose of finding biologically active of novel compounds and providing new ideas for drug-design, we performed virtual screening in commercially available databases and got pyrazoleanthrone with promising inhibitory activity against Aurora A. Optimization of solvent accessible C7 position of pyrazoleanthrone made us get thirteen target compounds. These pyrazoleanthrone derivatives were evaluated by Aurora A inhibition assays in vitro. The results show that some target compounds could inhibit Aurora A kinase. Meanwhile, these title compounds were tested in vitro against hepatocellular carcinoma（HepG2） cells by the 3＇-（4,5-dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide（MTT） method, showing that most of them had inhibitory potency. The inhibition rate of compound 6h was about 80% against HepG2 cells, and the 1C50 value was 17.4 μmol/L, which would be considered for further study.