A NMR and computational study of Smac mimics targeting both the BIR2 and BIR3 domains in XIAP protein

In this paper we report an extensive NMR analysis of small ligands (Smac mimics) complexed with different constructs of XIAP. The mimics-binding site of XIAP is known as the BIR3 domain - primary, and the linker BIR2 region - secondary site. Interactions between the BIR3 domain and Smac mimics have been extensively studied by X-ray but, as of today, there are scarce data about the interaction between BIR2, or the whole linker-BIR2-BIR3 construct, and Smac mimics. In order to characterize our Smac mimics, we performed a STD NMR study between our 4-substituted, 1-aza-2-oxobicyclo[5.3.0]decane scaffold-based molecules and three different XIAP fragments: single BIR2 and BIR3 domains, and bifunctional linker-BIR2-BIR3. The results were integrated with docking calculations and molecular dynamics simulations. NMR data, which are consistent with biological tests, indicated that the two BIR subunits interact differently with our Smac mimics and suggest that the ligands enter into more intimate contact with the linker-BIR2-BIR3. In conclusion, we observe that the SMAC mimics showed with the construct linker-BIR2-BIR3 a series of NOE contacts that were not observed in the mono-domain ligand:BIR2 or :BIR3 complexes. So, in agreement with the computational models we believe that the linker moieties of the binding site play a key role in the stability of the protein complex.     

Structure-Activity Relationship of NF023 Derivatives Binding to XIAP-BIR1

Inhibitors of Apoptosis Proteins (IAPs) are conserved E3-ligases that ubiquitylate substrates to prevent apoptosis and activate the NF-kB survival pathway, often deregulated in cancer. IAPs-mediated regulation of NF-kB signaling is based on the formation of protein complexes by their type-I BIR domains. The XIAP-BIR1 domain dimerizes to bind two TAB1 monomers, leading to downstream NF-kB activation. Thus, impairment of XIAP-BIR1 dimerization could represent a novel strategy to hamper cell survival in cancer. To this aim, we previously reported NF023 as a potential inhibitor of XIAP-BIR1 dimerization. Here we present a thorough analysis of NF023 binding to XIAP-BIR1 through biochemical, biophysical and structural data. The results obtained indicate that XIAP-BIR1 dimerization interface is involved in NF023 binding, and that NF023 overall symmetry and the chemical features of its central moiety are essential for an efficient interaction with the protein. Such strategy provides original hints for the development of novel BIR1-specific compounds as pro-apoptotic agents.     

Design, synthesis, and characterization of a potent, nonpeptide, cell-permeable, bivalent Smac mimetic that concurrently targets both the BIR2 and BIR3 domains in XIAP

XIAP is a central apoptosis regulator that inhibits apoptosis by binding to and inhibiting the effectors caspase-3/-7 and an initiator caspase-9 through its BIR2 and BIR3 domains, respectively. Smac protein in its dimeric form effectively antagonizes XIAP by concurrently targeting both its BIR2 and BIR3 domains. We report the design, synthesis, and characterization of a nonpeptide, cell-permeable, bivalent small-molecule (SM-164) which mimics Smac protein for targeting XIAP. Our study shows that SM-164 binds to XIAP containing both BIR domains with an IC50 value of 1.39 nM, being 300 and 7000 times more potent than its monovalent counterparts and the natural Smac AVPI peptide, respectively. SM-164 concurrently interacts with both BIR domains in XIAP and functions as an ultrapotent antagonist of XIAP in both cell-free functional and cell-based assays. SM-164 targets cellular XIAP and effectively induces apoptosis at concentrations as low as 1 nM in the HL-60 leukemia cell line. The potency of bivalent SM-164 in binding, functional, and cellular assays is 2-3 orders of magnitude higher than its corresponding monovalent Smac mimetics.     

An aporphine alkaloid from Nelumbo nucifera as an acetylcholinesterase inhibitor and the primary investigation for structure-activity correlations

N-methylasimilobine (1), a new-found strong acetylcholinesterase (AChE) inhibitor, along with two weakly active aporphine alkaloids, nuciferine (2) and nornuciferine (3) were separated from Nelumbo nucifera. N-methylasimilobine (1) inhibited 50% of AChE activity at the concentrations of 1.5?±?0.2?μg?mL(-1) when the standard IC(50) value of Physostigmine was 0.013?±?0.002?μg?mL(-1). The mode of AChE inhibition by 1 was reversible and non-competitive. In addition, molecular modelling was performed to explore the binding mode of inhibitor 1 at the active site of AChE.     

Role of p38 mitogen-activated protein kinase and caspases in UV-B-induced apoptosis of murine peritoneal macrophages

The mechanisms of ultraviolet-B (UV-B)-induced apoptosis and the role of p38 mitogen-activated protein kinase (MAPK) were investigated in murine peritoneal macrophages. Exposure of murine peritoneal macrophages to UV-B irradiation induced rapid apoptosis concurrent with DNA fragmentation and activation of caspase-3 but did not activate caspase-1. UV-B irradiation (100 mJ/cm2) also induced expression of phospho-p38 and -c-Jun N-terminal kinase (JNK) MAPK; however, no significant expression of phospho-p42/44 was observed 120 min after exposure. Pretreatment of macrophages with a p38 MAPK inhibitor, 4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)-1H-imidazole (SB202190), and a caspase-3 inhibitor, N-acetyl-Asp-Glu-Val-Asp-CHO, suppressed UV-B irradiation-induced apoptosis as observed by DNA laddering and DNA fragmentation estimation quantitatively. Pretreatment with caspase-1 inhibitor, N-acetyl-Tyr-Val-Ala-Asp-CHO, had no effect. UV-B-induced caspase-3 activation resulted in the cleavage of poly-(ADP-ribose) polymerase (PARP), which was inhibited by the caspase-3 inhibitor. SB202190 pretreatment also prevented activation of caspase-3 and the cleavage of PARP. However, the caspase-3 and -1 inhibitors did not affect UV-B-induced expression of phospho-p38 and -JNK. These results suggest that activation of p38 MAPK upstream of caspases might play an important role in the apoptotic process of macrophages exposed to UV-B irradiation.     

New anti-corrosion inhibitor (3ar,6ar)-3a,6a-di-p-tolyltetrahydroimidazo[4,5-d]imidazole-2,5(1 h,3h)-dithione for carbon steel in 1 M HCl medium: gravimetric,electrochemical, surface and quantum chemical analyses

The inhibition of (3ar,6ar)-3a,6a-di-p-tolyltetrahydroimidazo[4,5-d]imidazole-2,5(1 h,3h)-dithione (TTHIIDT) for carbon steel was full characterized in a 1 M hydrochloride acid environment at various inhibitor concentrations and temperatures by the gravimetric, electrochemical, surface and quantum chemical analyses. The obtained results confirmed that the inhibition efficiency of TTHIIDT was over 95–97% and nearly stable in the rise of temperature and concentration; TTHIIDT was mixed type inhibitor and effectively influenced both anodic and cathodic corrosion reactions; a protective hydrophobic thin layer of this inhibitor on the carbon steel surface is more stable and non soluble in 1 M HCl medium; this inhibitor adsorbed endothermically on the carbon steel surface by the chemical and physical adsorption processes. The quantum chemical calculations supported the experimental results and showed that the inhibition efficiency is depends on the structure of inhibitor.     

Adsorption,Surface Morphological,and Electrochemical Studies on the Inhibitive Properties of 4-(N,N-dimethylaminobenzilidine)-3-mercapto-6-methyl-1, 2, 4-triazin (4H)-5-one (DAMMT) on Mild Steel in 0.5 N H2SO4

Inhibition of mild steel corrosion in 0.5 N sulfuric acid by 4-(N, N-dimethylaminobenzilidine)-3-mercapto-6-methyl-1, 2, 4-triazin (4H)-5-one (DAMMT) was studied using polarization studies (Tafel); electrochemical impedance spectroscopy studies (EIS), adsorption studies, and surface morphological studies (SEM and AFM). The effect of inhibitor concentrations on corrosion rate, the effect of temperature, degree of surface coverage, adsorption kinetics, and surface morphology are investigated. Results show that the rate of corrosion increases with temperature in the absence and presence of inhibitor. Activation energies and enthalpies of activation in the presence and absence of DAMMT were obtained by measuring the temperature dependence of the corrosion current. DAMMT exhibits excellent inhibition properties towards MS and act as a mixed type inhibitor.     

Geissoschizine methyl ether, a corynanthean-type indole alkaloid from Uncaria rhynchophylla as a potential acetylcholinesterase inhibitor

Geissoschizine methyl ether (1), a newly discovered strong acetylcholinesterase (AChE) inhibitor, along with six weakly active alkaloids, vallesiachotamine (2), hisuteine (3), hirsutine (4), isorhynchophylline (5), cisocorynoxeine (6) and corynoxeine (7) have been isolated from Uncaria rhynchophylla. Geissoschizine methyl ether (1) inhibited 50% of AChE activity at concentrations of 3.7?±?0.3?μg?mL(-1) while the IC(50) value of physostigmine as a standard was 0.013?±?0.002?μg?mL(-1). The mode of AChE inhibition by 1 was reversible and non-competitive. In addition, molecular modelling was performed to explore the binding mode of inhibitor 1 at the active site of AChE.     

Synthesis and Antitumor Activity of a New 7-Azaindole Derivative

We designed and synthesized a 7-azaindole derivative（TH1082）, which was characterized by 1^H NMR and 13^C-NMR. We investigated its antitumor effects on human melanoma A375 cells, human liver cancer SMMC cells and human breast cancer MCF-7 cells in vitro via 3-（4,5-dimethyldiazol-2-yl）-2,5-diphenyl-tetrazolium bromide （MTT） assay and also explored the mechanism of antiproliferation of them. The results show that TH1082 significantly inhibited the proliferation of these cells to different extent. The IC50 values for A375 cells, SMMC cells and MCF-7 cells were 25.38, 48.70 and 76.94 μg/mL at 24 h, respectively. To observe cell morphological changes, acridine orange/ethidium bromide（AO/EB） staining and Hoechest33342/Pl staining were carried out. These results indicate that TH1082 could induced the apoptosis of A375 cells. The apoptotic rates were （9.5±2.09）%, （18.9±2.25）% and （39.5±2.02）%（5, 10 and 20 μg/mL） for A375, SMMC and MCF-7 cell lines, respectively. Further, we determined the activities of caspase-3 and caspase-9 in A375 cells treated with TH1082 at different concentrations（0, 5, 10 and 20 μg/mL） or Z-VAD-FMK（20 μmol/L）, a pan-caspase inhibitor for 24 h. The results show that TH1082 activated caspase-3 and caspase-9, and the activation could be blocked by Z-VAD-FMK. Taken together, these findings indicate that TH 1082 could inhibit the proliferation ofA375 cells via activating caspase-3 and caspase-9.     

氨基酸缓蚀剂在盐酸溶液中对钢的缓蚀作用

应用极化曲线、电化学阻抗谱等测试技术,研究了以谷氨酸作缓蚀剂在10%盐酸介质中对X65钢的缓蚀作用,测试腐蚀参数包括腐蚀电位(Ecorr)、极化电阻(Rp)、缓蚀效率(η)等.研究表明:该缓蚀剂是一种抑制阴极为主的混合型缓蚀剂,在较低的浓度具有良好缓蚀效果;缓蚀效率随着浓度的增加而提高,并在0.3g.L-1时达到最大值(90%以上).     

Corrosion inhibitive evaluation and DFT studies of 2-(Furan-2-yl)-4,5-diphenyl-1H-imidazole on mild steel at 1.0M HCl

A novel heterocyclic compound 2-(Furan-2-yl)-4,5-Diphenyl-1H-Imidazole (FDPI) was synthesized by a simple and cost effective one pot synthetic protocol and the structure of FDPI was confirmed by FT-IR, ¹H NMR and ¹³C NMR spectra. The corrosion inhibition activity of FDPI was investigated using gravimetric and electrochemical methods. It resulted a maximum inhibition efficiency of 95.84% at 10 mmolL⁻¹ concentrations of FDPI. The excellent inhibition efficiency is reasoned as the adsorption of FDPI on the mild steel surface as a protective layer immersed in the 1 ​M HCl. The adsorbed layer obeys Langmuir adsorption isotherm and the ΔG^o_ads values of FDPI suggested that process involves physisorption. The polarization curves showed that the FDPI behaves as a mixed type inhibitor. Surface morphology studied by SEM confirmed the formation of a protective film of FDPI on the mild steel surface. The computational studies using DFT have been analyzed for the FDPI to determine the HOMO-LUMO energy gap.     

The antiapoptotic effect of low-dose UVB irradiation in NIH3T3 cells involves caspase inhibitions

UVB irradiation is a well-known apoptosis induction factor. However, we have previously found that low doses of UVB irradiation inhibited apoptosis induced by both serum starvation and lack of extracellular matrix, involving a significant inhibition of caspase-3/7 activation. In this study, we report on the relationship between the UVB-induced anti-apoptotic effect and caspase-3/7 inhibition by reactive oxygen species (ROS). The UVB-induced antiapoptotic effect was partially prevented by an antioxidant agent, N-acetylcysteine. A ROS-generating agent, menadione and a pro-oxidant agent, H2O2 also showed an effect that was similar to the UVB-induced antiapoptotic effect, indicating that ROS contributed to the antiapoptotic effect. UVB irradiation significantly suppressed caspase-3/7 activation, which was caused by the inhibition of proteolysis and not by the inhibition of enzymatic activity itself. The prevention of proteolysis was also confirmed by both the following results: one is the inhibition of in vitro caspase-3/7 and -9 activation in cell lysates exposed to UVB in the presence of cytochrome c and dATP, which was caused by the production of ROS, and the other is the inhibition of in vitro caspase-3/7 activation in the presence of active caspase-9. These results showed that the inhibition of the caspase cascade downstream mitochondria by ROS production, leading to a significant inhibition of caspase-3/7 activation, was one of the causes of the antiapoptotic effect by small doses of UVB irradiation.     

The structures of caspases-1, -3, -7 and -8 reveal the basis for substrate and inhibitor selectivity

BACKGROUND: Peptide inhibitors of caspases have helped define the role of these cysteine proteases in biology. Structural and biochemical characterization of the caspase enzymes may contribute to the development of new drugs for the treatment of caspase-mediated inflammation and apoptosis. RESULTS: The crystal structure of the previously unpublished caspase-7 (Csp7; 2.35 A) bound to the reversible tetrapeptide aldehyde inhibitor acetyl-Asp-Glu-Val-Asp-CHO is compared with crystal structures of caspases-1 (2.3 A), -3 (2.2 A), and -8 (2.65 A) bound to the same inhibitor. Csp7 is a close homolog of caspase-3 (Csp3), and these two caspases possess some quarternary structural characteristics that support their unique role among the caspase family. However, although Csp3 and Csp7 are quite similar overall, they were found to have a significantly different substitution pattern of amino acids in and around the S4-binding site. CONCLUSIONS: These structures span all three caspase subgroups, and provide a basis for inferring substrate and inhibitor binding, as well as selectivity for the entire caspase family. This information will influence the design of selective caspase inhibitors to further elucidate the role of caspases in biology and hopefully lead to the design of therapeutic agents to treat caspase-mediated diseases, such as rheumatoid arthritis, certain neurogenerative diseases and stroke.     

Effects of BAPTA-AM and Forskolin on Apoptosis and Cytochrome c Release in Photosensitized Chinese Hamster V79 Cells

The mechanism of caspase-3-dependent apoptosis induced by photodynamic therapy (PDT) of cultured Chinese hamster V79 cells with pheophorbide a (PPa) was investigated. The PPa-PDT induced rapid apoptosis within 30 min after irradiation of cells. This apoptosis was inhibited by the 1O2 quencher N3- and caspase-3 inhibitor acetyl-Asp-Glu-Val-Asp-aldehyde, suggesting that 1O2 activated caspase-3 and then caused apoptosis. The intracellular calcium [Ca2+]i chelator (acetoxymethyl)-1,2-bis(o-aminophenoxy)ethane N,N,N',N'-tetraacetic acid (BAPTA-AM) and the cyclic adenosine monophosphate (cAMP)-increasing agent forskolin also inhibited not only the PPa-PDT-induced activation of caspase-3 but also apoptosis in V79 cells. Furthermore, PPa-PDT-induced cytochrome c release from mitochondria was found to be inhibited by the treatment with BAPTA-AM but not forskolin. These results indicated that [Ca2+]i and cAMP independently serve as regulators for PPa-PDT-induced apoptosis in the upstream of caspase-3.     

Inhibition of Mild Steel Corrosion in 1 M Hydrochloric Acid Using (E)-(4-(4-methoxybenzylideneamino)-4H-1,2,4-triazole-3,5-diyl) Dimethanol (MBATD)

The inhibition of mild steel corrosion in 1 M HCl by (E)-(4-(4-methoxybenzylideneamino)-4H-1, 2,4-triazole-3,5-diyl) dimethanol (MBATD) was investigated by polarization, AC impedance, thermodynamic, molecular dynamics, and quantum chemical studies. Polarization studies revealed that MBATD act as mixed type inhibitor. Adsorption followed the Langmuir mode with a negative value of free energy, which indicates a stable and spontaneous inhibition process. To understand the energy changes associated with various thermodynamic and kinetic processes, different calculations were made. The correlation between inhibitive action and molecular structure is ascertained through quantum chemical calculations. The adsorption behavior of the inhibitor molecule on mild steel surface has been theoretically computed using molecular dynamics and density functional theory.     

Vitexin inhibits polyubiquitin synthesis by the ubiquitin-conjugating enzyme E2-25K

An extract of bark from the tropical rainforest plant Byrsonima crassifolia was screened for inhibition of diubiquitin formation by the human ubiquitin-conjugating enzyme E2-25K. Activity assays with both the full-length enzyme and a truncated, active catalytic UBC domain revealed that the extract contained inhibitory properties. Separation of the extract into individual components and additional screens identified vitexin as the active inhibitor. An IC50 for vitexin was calculated to be approximately 0.5 mM. Molecular modeling simulations were used to predict the mode of inhibition and NMR spectra were used to confirm the binding site of vitexin to E2-25K.     

Influence of 5-chloro and 5-methyl benzotriazole on the corrosion of copper in acid solution: an experimental and a theoretical approach

The inhibition of copper corrosion in aerated 0.1 mol l⁻¹ hydrochloric acid solutions was studied using electrochemical polarization in the presence of different concentrations of benzotriazole and its two derivatives, 5-chloro and 5-methyl benzotriazole. The inhibition efficiencies obtained from cathodic Tafel plots increased markedly with increase in the additive concentration. Benzotriazole and 5-methyl-benzotriazole were found to be cathodic type corrosion inhibitors for concentrations higher than 10⁻⁴ mol l⁻¹ . However, the 5-chloro-benzotriazole was found to be a mixed inhibitor for concentrations up to 10⁻³ mol l⁻¹, above this concentration the inhibitor behaves as an anodic type inhibitor. The inhibitors are physisorbed on the copper surface following a Langmuir’s isotherm. The inhibition efficiencies depended on the inhibitor concentration and follows the order 5-chloro-benzotriazole > 5-methyl-benzotriazole > 1-H-benzotriazole. From the theoretical calculations, the change in the inhibition mechanism observed for 5-chloro-benzotriazole at concentrations higher than 10⁻³ mol l⁻¹ is associated with the electronic acceptor characteristic of chloro, which increases the benzotriazole acidity allowing the formation of CuBTA.     

Corrosion inhibition of 302 stainless steel with schiff base compounds

The effects of 2,2′-[bis-N(4-cholorobenzaldimin)]-1,1′-dithio (BCBD) and bis-(2-aminophenyl) disulphide (BAPD) on the corrosion behavior of 302 stainless steel in 0.5 M sulfuric acid solution as corrosive medium were investigated using weight loss and potentiostatic polarization techniques. Some corrosion parameters such as anodic and cathodic Tafel slopes, corrosion potential, corrosion current density, surface coverage degrees and inhibition efficiencies were calculated. The polarization measurements indicated that the inhibitors were of mixed type which inhibited corrosion by parallel adsorption on the surface of stainless steel due to the presence of more than one active centre in the inhibitor molecule. The adsorption followed Langmuir adsorption isotherm. The activation energy and thermodynamic parameters were calculated at different temperatures. Results showed that BCBD had a higher inhibition efficiency compared with BAPD.