Lipoxygenase inhibitor from Lactobacillus casei

An inhibitor of plant lipoxygenase from culture filtrates of Lactobacillus casei was purified by column chromatography and shown to be benzoic acid. The isolated benzoic acid had an IC₅₀ of 350 M against purified soybean lipoxygenase at pH 9. L. casei therefore may have the potential to be used as a preservative against the oxidation of unsaturated fatty acids, thereby preventing undesirable flavours in foods.     

Methylene substitution at the alpha-beta bridging position within the phosphate chain of dUDP profoundly perturbs ligand accommodation into the dUTPase active site

dUTP pyrophosphatase, a preventive DNA repair enzyme, contributes to maintain the appropriate cellular dUTP/dTTP ratio by catalyzing dUTP hydrolysis. dUTPase is essential for viability in bacteria and eukaryotes alike. Identification of species-specific antagonists of bacterial dUTPases is expected to contribute to the development of novel antimicrobial agents. As a first general step, design of dUTPase inhibitors should be based on modifications of the substrate dUTP phosphate chain, as modifications in either base or sugar moieties strongly impair ligand binding. Based on structural differences between bacterial and human dUTPases, derivatization of dUTP-analogous compounds will be required as a second step to invoke species-specific character. Studies performed with dUTP analogues also offer insights into substrate binding characteristics of this important and structurally peculiar enzyme. In this study, alpha,beta-methylene-dUDP was synthesized and its complex with dUTPase was characterized. Enzymatic phosphorylation of this substrate analogue by pyruvate kinase was not possible in contrast to the successful enzymatic phosphorylation of alpha,beta-imino-dUDP. One explanation for this finding is that the different bond angles and the presence of the methylene group may preclude formation of a catalytically competent complex with the kinase. Crystal structure of E. coli dUTPase:alpha,beta-methylene-dUDP and E. coli dUTPase:dUDP:Mn complexes were determined and analyzed in comparison with previous data. Results show that the "trans" alpha-phosphate conformation of alpha,beta-methylene-dUDP differs from the catalytically competent "gauche" alpha-phosphate conformation of the imino analogue and the oxo substrate, manifested in the shifted position of the alpha-phosphorus by more than 3 A. The three-dimensional structures determined in this work show that the binding of the methylene analogue with the alpha-phosphorus in the "gauche" conformation would result in steric clash of the methylene group with the protein atoms. In addition, the metal ion cofactor was not bound in the crystal of the complex with the methylene analogue while it was clearly visible as coordinated to dUDP, arguing that the altered phosphate chain conformation also perturbs metal ion complexation. Isothermal calorimetry titrations indicate that the binding affinity of alpha,beta-methylene-dUDP toward dUTPase is drastically decreased when compared with that of dUDP. In conclusion, the present data suggest that while alpha,beta-methylene-dUDP seems to be practically nonhydrolyzable, it is not a strong binding inhibitor of dUTPase probably due to the altered binding mode of the phosphate chain. Results indicate that in some cases methylene analogues may not faithfully reflect the competent substrate ligand properties, especially if the methylene hydrogens are in steric conflict with the protein.     

A new type of pharmacological chaperone for GM1-gangliosidosis related human lysosomal β-galactosidase: N-Substituted 5-amino-1-hydroxymethyl-cyclopentanetriols

N-Functionalized amino(hydroxymethyl)cyclopentanetriols are potent inhibitors of β-d-galactosidases and, for the first time, could be shown to act as pharmacological chaperones for G_M1-gangliosidosis-associated lysosomal acid β-galactosidase thus representing a new structural type of pharmacological chaperones for this lysosomal storage disease.     

Discovery of 5-substituent-N-arylbenzamide derivatives as potent,selective and orally bioavailable LRRK2 inhibitors

Leucine-rich repeat kinase 2 (LRRK2) has been suggested as a potential therapeutic target for Parkinson’s disease. Herein we report the discovery of 5-substituent-N-arylbenzamide derivatives as novel LRRK2 inhibitors. Extensive SAR study led to the discovery of compounds 8e, which demonstrated potent LRRK2 inhibition activity, high selectivity across the kinome, good brain exposure, and high oral bioavailability.     

Structure-activity relationships and molecular docking of thirteen synthesized flavonoids as horseradish peroxidase inhibitors

For the first time, the structure–activity relationships of thirteen synthesized flavonoids have been investigated by evaluating their ability to modulate horseradish peroxidase (HRP) catalytic activity. Indeed, a modified spectrophotometrically method was carried out and optimized using 4-methylcatechol (4-MC) as peroxidase co-substrate.The results show that these flavonoids exhibit a great capacity to inhibit peroxidase with Ki values ranged from 0.14 ± 0.01 to 65 ± 0.04 mM. Molecular docking has been achieved using Auto Dock Vina program to discuss the nature of interactions and the mechanism of inhibition. According to the docking results, all the flavonoids have shown great binding affinity to peroxidase. These molecular modeling studies suggested that pyran-4-one cycle acts as an inhibition key for peroxidase. Therefore, potent peroxidase inhibitors are flavonoids with these structural requirements: the presence of the hydroxyl (OH) group in 7, 5 and 4′ positions and the absence of the methoxy (O–CH₃) group. Apigenin contributed better in HRP inhibitory activity.The present study has shown that the studied flavonoids could be promising HRP inhibitors, which can help in developing new molecules to control thyroid diseases.     

Evidence that the Modulation of Membrane-Associated Protein Kinase C Activity by an Endogenous Inhibitor Plays a Role in N1E-115 Murine Neuroblastoma Cell Differentiation

Abstract: Murine neuroblastoma cells, N1E-115, were induced to differentiate into neuron-like cells by serum deprivation for 18 h. As previous studies have shown that the suppression of protein kinase C (PKC) activity by selective inhibitors or neutralizing antibodies induces neuroblastoma cells to differentiate, we tested the hypothesis that serum deprivation may cause a rapid loss in membrane PKC activity that occurs well before the morphological changes that are characteristic of cell differentiation. A significant reduction in particulate (membrane) PKC activity was indeed observed within 3 h of serum withdrawal when enzyme activity was measured in intact native membranes by the recently described in vitro "direct" assay. This rapid reduction in enzyme activity was confirmed by the decreased phosphorylation of the MARCKS protein, an endogenous PKC-selective substrate, in intact cells. The decrease in membrane PKC activity occurred without any loss in the amount of membrane-associated enzyme, suggesting that some factor(s) resident in neuroblastoma membranes was suppressing PKC activity. Indeed, results indicate the presence of an endogenous inhibitor of PKC tightly associated with neuroblastoma membranes. This inhibitory activity increased in the membranes of cells subjected to serum deprivation, raising the possibility that it was likely responsible for the decline in membrane PKC activity in differentiating N1E-115 cells. Preliminary characterization indicated that the inhibitory activity is a protein and is localized mainly in the membrane fraction. Thus, these results demonstrate directly that endogenous inhibitor can regulate membrane-associated PKC activity in cells and thereby modulate PKC-related neuronal functions.     

Three-dimensional structure of the complex of 4-guanidino-Neu5Ac2en and influenza virus neuraminidase.

The three-dimensional X-ray structure of a complex of the potent neuraminidase inhibitor 4-guanidino-Neu5Ac2en and influenza virus neuraminidase (Subtype N9) has been obtained utilizing diffraction data to 1.8 A resolution. The interactions of the inhibitor, solvent water molecules, and the active site residues have been accurately determined. Six water molecules bound in the native structure have been displaced by the inhibitor, and the active site residues show no significant conformational changes on binding. Sialic acid, the natural substrate, binds in a half-chair conformation that is isosteric to the inhibitor. The conformation of the inhibitor in the active site of the X-ray structure concurs with that obtained by theoretical calculations and validates the structure-based design of the inhibitor. Comparison of known high-resolution structures of neuraminidase subtypes N2, N9, and B shows good structural conservation of the active site protein atoms, but the location of the water molecules in the respective active sites is less conserved. In particular, the environment of the 4-guanidino group of the inhibitor is strongly conserved and is the basis for the antiviral action of the inhibitor across all presently known influenza strains. Differences in the solvent structure in the active site may be related to variation in the affinities of inhibitors to different subtypes of neuraminidase.     

Characterization of a growth-inhibiting protein present in rat serum that exerts a differential effect onin vitro growth of nonmalignant rat liver cells when compared with Rous sarcoma virus-transformed rat liver cells

Summary We have previously reported the transformation by Rous sarcoma virus of a cloned epithelial cell line (BRL) established from Buffalo rat liver by H. Coon. The nontransformed (BRL) and transformed (RSV-BRL) cells grew at comparable rates in culture, whereas only the transformed cells were tumorigenic in vivo. We report here on the existence in rat and mouse sera of a growth inhibitor for the nontransformed BRL cells. The transformed BRL cells (RSV-BRL) were insensitive to this inhibitor. The inhibitory activity was not prominent in sera from other species of animals tested except for rabbit; this serum inhibited the growth of RSV-BRL cells more strongly than that of BRL cells. The growth inhibitor was partially purified from rat serum. It is a protein free of lipid and has a molecular weight of about 220 000. The inhibitor could be separated into three components of pI 4.6, 5.2 (major) and 5.6 by isoelectric electrophoresis. EDITOR'S STATEMENT Although compelling theoretical arguments sometimes can be made for the likely existence of growth-inhibitory substances of physical relevance in the control of cell proliferation, experiments aimed at identifying and studying such factors often are difficult to design and interpret, and little strong data exists to suggest that growth-inhibitory substances are important regulatorsin vivo. The information presented in this paper represents a start toward developing a useful system for studying growth-inhibitory factor. David W. Barnes     

Sucrose metabolism during endosperm development in wheat (Triticum aestivum)

This work reports changes in sucrose synthase and invertase activities throughout endosperm development in wheat, together with the associated substrates and metabolites, sucrose, UDP, glucose, fructose and UDP-glucose. Throughout endosperm development, sucrose synthase had consistently higher activity than invertase and indeed invertase activity did not change appreciably. The observed variation in pattern and amounts of glucose and fructose present during the mid- and late stages of endosperm development confirmed the suggestion that invertase was not the preferred pathway of sucrose catabolism. Kinetic parameters for sucrose synthase were determined in crude extracts. Estimates of UDP and sucrose concentrations suggest that sucrose synthase is unlikely to achieve its potential maximum velocity. This limitation may however be overcome in part by the apparent excess catalytic activity measured during endosperm development.     

The pan-ErbB receptor tyrosine kinase inhibitor canertinib promotes apoptosis of malignant melanoma in vitro and displays anti-tumor activity in vivo

The ErbB receptor family has been suggested to constitute a therapeutic target for tumor-specific treatment of malignant melanoma. Here we investigate the effect of the pan-ErbB tyrosine kinase inhibitor canertinib on cell growth and survival in human melanoma cells in vitro and in vivo. Canertinib significantly inhibited growth of cultured melanoma cells, RaH3 and RaH5, in a dose-dependent manner as determined by cell counting. Half-maximum growth inhibitory dose (IC₅₀) was approximately 0.8 μM and by 5 μM both cell lines were completely growth-arrested within 72 h of treatment. Incubation of exponentially growing RaH3 and RaH5 with 1 μM canertinib accumulated the cells in the G₁-phase of the cell cycle within 24 h of treatment without induction of apoptosis as determined by flow cytometry. Immunoblot analysis showed that 1 μM canertinib inhibited ErbB1-3 receptor phosphorylation with a concomitant decrease of Akt-, Erk1/2- and Stat3 activity in both cell lines. In contrast to the cytostatic effect observed at doses ?5 μM canertinib, higher concentrations induced apoptosis as demonstrated by the Annexin V method and Western blot analysis of PARP cleavage. Furthermore, canertinib significantly inhibited growth of RaH3 and RaH5 melanoma xenografts in nude mice. Pharmacological targeting of the ErbB receptors may prove successful in the treatment of patients with metastatic melanoma.