Dibasic benzo[B]thiophene derivatives as a novel class of active site directed thrombin inhibitors: 4. SAR studies on the conformationally restricted C3-side chain of hydroxybenzo[B]thiophenes

A novel series of benzo[b]thiophene diamine thrombin inhibitors with a conformationally restricted C3-side chain 3 was investigated. The constrained C3-side chain by a cyclohexyl ring contributed to not only an additive but also a synergistic effect on the thrombin inhibitory activity. The SAR studies resulted in the discovery of a potent thrombin inhibitor 27 that was over 750-fold more potent than the initial lead compound 1.     

Cytochromes and hydrogen-oxidizing activity in the thermophilic hydrogen-oxidizing bacteria related to the genus Hydrogenobacter

The highly thermophilic, hydrogen-oxidizing aerobic bacteria related to Hydrogenobacter possess a respiratory chain comprising a quinone and b-type (alpha band at 556 nm and 562 nm) and c-type (alpha band at 552 nm) cytochromes. They have no aa₃-type cytochromes and their terminal oxidase is an o-type cytochrome. A polarographic method with an oxygen electrode was used for the measurement of the hydrogen-oxidizing activity. This activity was strongly inhibited by HQNO (2-N-heptyl-4-hydroxyquinoline N-oxide), an inhibitor of the respiratory chain in the quinone-cytochrome b region, and by KCN, an inhibitor of the terminal cytochrome oxidase. This study shows that the electrons released from hydrogen oxidation by the membrane-bound hydrogenase probably enter the respiratory chain at the level of the quinone-cytochrome b region.Abbreviations HQNO 2-N-heptyl-4-hydroxyquinoline N-oxide - TMPD N,N,N',N'-tetramethyl-p-phenylenediamine - DW dry weight     

Chemical structure of two fragments of human serum albumin and their location in the albumin molecule.

1. 'Inhibitor fragment' isolated from human serum albumin degraded by rabbit cathepsin D is composed of one peptide chain with two intrachain disulphide bonds. There are two kinds of inhibitor molecules having different N-terminal amino acids: one is threonine and the other glutamine. 2. Fragment F1, isolated from inhibitor degraded by trypsin, is composed of two chains linked by a disulphide bond. There are three kinds of fragment F1. All have one alpha chain in common, which has an intrachain disulphide bond. They differ by the nature of the chain, which is linked to the alpha chain by a disulphide bond. The epsilon chain is present in trace amounts. The two other chains, beta and gamma, differ by their C-terminal amino acid, which is respectively arginine and lysine. 3. Inhibitor is composed of the last 92 or 89 residues of the human albumin molecule and fragment F1 is composed of two parts of this C-terminal portion of the albumin molecule.     

Study of the insulin signaling pathways in the regulation of ACAT1 expression in cultured macrophages

Objective

To determine the signaling pathways and components involved in insulin-mediated regulation of Acyl-CoA: cholesterol acyltransferase1 (ACAT1).

Methods

THP-1 cells were cultured in RPMI 1640 medium and were induced into macrophages in the presence of 160 nM phorbol 12-myristate 13-acetate (PMA). Before insulin was added in, macrophages were preincubated with the inhibitors of the insulin signaling pathway, including wortmannin, phosphatidylinositol 3-kinase (PI3 K) inhibitor; PD98059, extracellular signal-regulated kinase (ERK) inhibitor; SB203580, p38 mitogen-activated protein kinase (p38MAPK) inhibitor; SP600125, c-Jun N-terminal kinase (JNK) inhibitor and U73122, phospholipase C-γ (PLC-γ) inhibitor. ACAT1 mRNA and protein expression level in macrophages were determined by real-time quantitative polymerase chain reaction and western blotting, respectively.

Results

Real-time quantitative polymerase chain reaction and western blotting demonstrated that PD98059, SB203580 or SP600125 down-regulated the expression of ACAT1 in a dose-dependent manner. However, no obvious alteration was found in wortmannin and U73122 groups.

Conclusion

These results suggest that the ERK, p38MAPK and JNK signaling pathways may be involved in insulin-mediated regulation of ACAT1, but no PI3K and PLC-γ signaling pathways were involved in the present study.     

Alpha-1-C-alkyl-1-deoxynojirimycin derivatives as potent and selective inhibitors of intestinal isomaltase: remarkable effect of the alkyl chain length on glycosidase inhibitory profile

A series of - and β-1-C-alkyl-1-deoxynojirimycin derivatives was prepared and evaluated as glycosidase inhibitors. Biological assays showed a marked dependence of the selectivity and potency of the inhibitors upon the position of the alkyl chain (-1-C-, β-1-C- or N-alkyl derivatives). In addition, the efficiency of -1-C-alkyl-1-deoxynojirimycin derivatives as intestinal isomaltase inhibitors increases with the length of the alkyl chain. The strongest inhibition was found for -1-C -nonyl-1-deoxynojirimycin with an IC₅₀ = 3.5 nM (25× more potent inhibitor than the shorter chain homologue carrying a C₈ chain). These results demonstrate that subtle changes in the aglycon fragment may result in remarkable enzyme specificity.     

Isolation and characterization of hemorrhagic factors a and b from the venom of the Chinese habu snake (Trimeresurus mucrosquamatus)

Hemorrhagic factors a and b were isolated from Trimeresurus mucrosquamatus venom by Sephadex G-100, CM-Sephadex C-50 and DEAE-Sephacel column chromatographies. The hemorrhagic factors were homogeneous, as established by a single band on acrylamide gel electrophoresis, isoelectric focusing and sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Molecular weights of 15 000 and 27 000 were found for hemorrhagic factors a and b, respectively. Factor a possesses proteolytic activity hydrolyzing the His(10)-Leu(11), Tyr(16)-Leu(17) and Arg(22)-Gly(23) bonds of oxidized insulin B chain, whereas, factor b hydrolyzed only the Ala(14)-Leu(15) bond. Hemorrhagic activity of these hemorrhagic factors was inhibited by ethylenediaminetetraacetic acid, 1,10-phenanthroline or p-chloromercuribenzoate, but not by soybean trypsin inhibitor or diisopropyl fluorophosphate. The hemorrhagic factors were injected into the skin of the back of albino rabbits, and the minimum hemorrhagic dose of factors a and b was 1.7 and 2.3 micrograms, respectively. These purified hemorrhagic factors were not lethal at 15 micrograms/g in mice. Factor a hydrolyzed the B beta chain of fibrinogen, while factor b hydrolyzed the A alpha chain. Hemorrhagic factor a was shown to differ immunologically from factor b. Factors a and b produced systemic hemorrhage in internal organs such as the heart and stomach of mice. Moreover, factor b produced hemorrhage in the liver.     

Analysis of Binding Sites on Complement Factor I That Are Required for Its Activity

The central complement inhibitor factor I (FI) degrades activated complement factors C4b and C3b in the presence of cofactors such as C4b-binding protein, factor H, complement receptor 1, and membrane cofactor protein. FI is a serine protease composed of two chains. The light chain comprises the serine protease domain, whereas the heavy chain contains several domains; that is, the FI and membrane attack complex domain (FIMAC), CD5, low density lipoprotein receptor 1 (LDLr1) and LDLr2 domains. To understand better how FI acts as a complement inhibitor, we used homology-based models of FI domains to predict potential binding sites. Specific amino acids were then mutated to yield 16 well expressed mutants, which were then purified from media of eukaryotic cells for functional analyses. The Michaelis constant (K_m) of all FI mutants toward a small substrate was not altered, whereas some mutants showed increased maximum initial velocity (V_max). All the mutations in the FIMAC domain affected the ability of FI to degrade C4b and C3b irrespective of the cofactor used, whereas only some mutations in the CD5 and LDLr1/2 domains had a similar effect. These same mutants also showed impaired binding to C3met. In conclusion, the FIMAC domain appears to harbor the main binding sites important for the ability of FI to degrade C4b and C3b.     

Superoxide production and electron transport in mitochondrial oxidation of dihydroorotic acid.

Production of superoxide radical during oxidation of dihydroorotate in rat liver mitochondria was not affected by antimycin A, thenoyltrifluoroacetone, or added ubiquinone but was inhibited by orotate, a product inhibitor of dihydroorotate dehydrogenase. It appears likely that superoxide is generated at the primary dehydrogenase. Dihydroorotate dehydrogenase differs from succinate dehydrogenase both in its utilization of ubiquinone and in the mechanism of cytochrome b reduction. Thenoyltrifluoroacetone completely inhibits fumarate synthesis and reduction of cytochrome b by succinate. Formation of orotate is only partially inhibited by thenolytrifluoroacetone and the inhibitor does not prevent reduction of cytochrome b by dihydroorotate. It is proposed that several pathways exist for linkage of the primary dihydrorotate dehydrogenase with the electron transport chain. One route involves electron transfer from ubiquinone to cytochrome c and is inhibited by thenoyltrifluoroacetone. A second route bypasses ubiquinone and is inhibited by antimycin A. A third pathway utilizes both ubiquinone and cytochrome b and is partiayly inhibited by either thenoyltrifluoroacetone or antimycin A.     

Laminin α1 Chain Synthesis in the Mouse Developing Lung: Requirement for Epithelial–Mesenchymal Contact and Possible Role in Bronchial Smooth muscle Development

Laminins, the main components of basement membranes, are heterotrimers consisting of α, β, and γ polypeptide chains linked together by disulfide bonds. Laminins-1 and -2 are both composed of β1 and γ1 chains and differ from each other on their α chain, which is α1 and α2 for laminin-1 and -2, respectively. The present study shows that whereas laminins-1 and -2 are synthesized in the mouse developing lung and in epithelial–mesenchymal cocultures derived from it, epithelial and mesenchymal monocultures lose their ability to synthesize the laminin α1 chain. Synthesis of laminin α1 chain however returns upon re-establishment of epithelial–mesenchymal contact. Cell–cell contact is critical, since laminin α1 chain is not detected in monocultures exposed to coculture-conditioned medium or in epithelial–mesenchymal cocultures in which heterotypic cell–cell contact is prevented by an interposing filter. Immunohistochemical studies on cocultures treated with brefeldin A, an inhibitor of protein secretion, indicated both epithelial and mesenchymal cells synthesize laminin α1 chain upon heterotypic cell– cell contact. In a set of functional studies, embryonic lung explants were cultured in the presence of monoclonal antibodies to laminin α1, α2, and β/γ chains. Lung explants exposed to monoclonal antibodies to laminin α1 chain exhibited alterations in peribronchial cell shape and decreased smooth muscle development, as indicated by low levels of smooth muscle α actin and desmin. Taken together, our studies suggest that laminin α1 chain synthesis is regulated by epithelial–mesenchymal interaction and may play a role in airway smooth muscle development.     

Modulation of the activity of histone acetyltransferases by long chain alkylidenemalonates (LoCAMs)

A novel class of KAT modulators (long chain alkylidenemalonates, LoCAMs) has been identified. Variations of the alkyl chain length can change the activity profile from inhibition of both KAT3A/KAT2B (as derivative 2a) to the peculiar profile of pentadecylidenemalonate 1b, the first activator/inhibitor of histone acetyltransferases. Together with the powerful apoptotic effect (particularly notable if considering that anacardic acid and other KAT inhibitors are not cell permeable) appoint them as valuable biological tools to understand the mechanisms of lysine acetyltransferases.     

t-Butyl-4-hydroxyanisole, a novel respiratory chain inhibitor. Effects on Trypanosoma cruzi epimastigotes

t-Butyl-4-hydroxyanisole, an antioxidant food additive, inhibited the growth of Trypanosoma cruzi by almost 100% at 0.5 mM concentration. This compound inhibited 70% of oxygen consumption of epimastigotes. The redox level of NAD(P) was shifted to a more reduced state and inversely the redox level of cytochrome b changed to a more oxidized state. This hydroxyanisole thus is a new electron transport chain inhibitor. This compound and related ones, or the respiratory chain of T. cruzi, may be important in the design of antichagasic drugs.     

MicroRNA-33b regulates sensitivity to daunorubicin in acute myelocytic leukemia by regulating eukaryotic translation initiation factor 5A-2

In this study, we aimed to study the effect of miR-33b in regulating sensitivity to daunorubicin (DNR) in acute myelocytic leukemia (AML). We used quantitative real-time polymerase chain reaction and Cell Counting Kit-8 assay to detect the level of miR-33b and cell viability. Cell apoptosis and the expression of eIF5A-2 and MCL-1 protein were detected by flow cytometry analysis and Western Blot analysis, respectively. MiR-33b mimic increased sensitivity of AML cells against DNR, while miR-33b inhibitor had the opposite effect. Furthermore, the results showed that the eIF5A-2 gene was a direct target of miR-33b, and miR-33b regulated eIF5A-2 mRNA and protein expression. Silencing of eIF5A-2 by RNA interference increased the sensitivity of AML cells against DNR. We also found that MCL-1 contributed to the regulation of DNR sensitivity, which was dependent on downregulation of eIF5A-2. Finally, knockdown of eIF5A-2 eliminated the effects of miRNA-33b mimic or inhibitor on DNR sensitivity. These findings indicate that miR-33b maybe as a new therapeutic target in AML cells.     

Isolation and characterization of hemorrhagic factors a and b from the venom of the Chinese habu snake (Trimeresurus mucrosquamatus)

Hemorrhagic factors a and b were isolated from Trimeresurus mucrosquamatus venom by Sephadex G-100, CM-Sephadex C-50 and DEAE-Sephacel column chromatographies. The hemorrhagic factors were homogeneous, as established by a single band on acrylamide gel electrophoresis, isoelectric focusing and sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Molecular weights of 15 000 and 27 000 were found for hemorrhagic factors a and b, respectively. Factor a possesses proteolytic activity hydrolyzing the His(10)-Leu(11), Tyr(16)-Leu(17) and Arg(22)-Gly(23) bonds of oxidized insulin B chain, whereas, factor b hydrolyzed only the Ala(14)-Leu(15) bond. Hemorrhagic activity of these hemorrhagic factors was inhibited by ethylenediaminetetraacetic acid, 1,10-phenanthroline or p-chloromercuribenzoate, but not by soybean trypsin inhibitor or diisopropyl fluorophosphate. The hemorrhagic factors were injected into the skin of the back of albino rabbits, and the minimum hemorrhagic dose of factors a and b was 1.7 and 2.3 μg, respectively. These purified hemorrhagic factors were not lethal at 15 μg/g in mice. Factor a hydrolyzed the Bβ chain of fibrinogen, while factor b hydrolyzed the Aα chain. Hemorrhagic factor a was shown to differ immunologically from factor b. Factors a and b produced systemic hemorrhage in internal organs such as the heart and stomach of mice. Moreover, factor b produced hemorrhage in the liver.     

The Respiratory Chain of Plant Mitochondria. I. Electron Transport Between Succinate and Oxygen in Skunk Cabbage Mitochondria

The kinetics of oxidation of ubiquinone, flavoprotein, cytochrome c, and the cytochrome b complex in skunk cabbage (Symplocarpus foetidus) mitochondria made anaerobic with succinate have been measured spectrophotometrically and fluorimetrically in the absence of respiratory inhibitor and in the presence of cyanide or antimycin A. No component identifiable by these means was oxidized rapidly enough in the presence of one or the other inhibitor to qualify for the role of alternate oxidase. Cycles of oxidation and rereduction of flavoprotein and ubiquinone obtained by injecting 12 mum oxygen into the anaerobic mitochondrial suspension were kinetically indistinguishable in the presence of cyanide or antimycin A, implying that these 2 components are part of a respiratory pathway between succinate and oxygen which does not involve the cytochromes and does involve a cyanide-insensitive alternate oxidase. The cytochrome b complex shows biphasic oxidation kinetics with half times of 0.018 sec and 0.4 sec in the absence of inhibitor, which increase to 0.2 sec and 1 sec in the presence of cyanide. In the presence of antimycin A, the oxidation of the cytochrome b complex shows an induction period of 1 sec and a half-time of 3.5 sec. A split respiratory chain with 2 terminal oxidases and a branch point between the cytochromes and flavoprotein and ubiquinone is proposed for these mitochondria.     

Glycosylation causes an apparent block in translation of immunoglobulin heavy chain

Analysis of nascent heavy chains isolated from MPC11 (gamma 2b heavy chains) and MOPC 21 (gamma 1 heavy chains) mouse myeloma cells demonstrates an accumulation of nascent heavy chains which are slightly smaller in mass (approximately 35,000 daltons) than nascent heavy chains which have just been glycosylated (approximately 38,000 daltons). The accumulation of 35,000-dalton nascent heavy chain appears to be a consequence of the glycosylation process since tunicamycin, an inhibitor of glycosylation, abolishes the apparent translational block manifested by the accumulation of 35,000-dalton nascent chains. Tunicamycin also causes a 15 to 25% increase n the relative rate of synthesis of heavy chain compared to the corresponding rate of synthesis of the nonglycosylated light chain synthesized by the same cell. These results suggest that the translation block, caused by the glycosylation process, of heavy chain synthesis contributes to the imbalance of heavy chain and light chain biosynthesis observed in malignant and normal lymphoid cells.     

Identification of retinoic acid in a high content screen for agents that overcome the anti-myogenic effect of TGF-beta-1

Background

Transforming growth factor beta 1 (TGF-β1) is an inhibitor of muscle cell differentiation that is associated with fibrosis, poor regeneration and poor function in some diseases of muscle. When neutralizing antibodies to TGF-β1 or the angiotensin II inhibitor losartan were used to reduce TGF-β1 signaling, muscle morphology and function were restored in mouse models of Marfan Syndrome and muscular dystrophy. The goal of our studies was to identify additional agents that overcome the anti-myogenic effect of TGF-β1.

Methodology/Principal Findings

A high-content cell-based assay was developed in a 96-well plate format that detects the expression of myosin heavy chain (MHC) in C2C12 cells. The assay was used to quantify the dose-dependent responses of C2C12 cell differentiation to TGF-β1 and to the TGF-β1 Type 1 receptor kinase inhibitor, SB431542. Thirteen agents previously described as promoting C2C12 differentiation in the absence of TGF-β1 were screened in the presence of TGF-β1. Only all-trans retinoic acid and 9-cis retinoic acid allowed a maximal level of C2C12 cell differentiation in the presence of TGF-β1; the angiotensin-converting enzyme inhibitor captopril and 10 nM estrogen provided partial rescue. Vitamin D was a potent inhibitor of retinoic acid-induced myogenesis in the presence of TGF-β1. TGF-β1 inhibits myoblast differentiation through activation of Smad3; however, retinoic acid did not inhibit TGF-β1-induced activation of a Smad3-dependent reporter gene in C2C12 cells.

Conclusions/Significance

Retinoic acid alleviated the anti-myogenic effect of TGF-β1 by a Smad3-independent mechanism. With regard to the goal of improving muscle regeneration and function in individuals with muscle disease, the identification of retinoic acid is intriguing in that some retinoids are already approved for human therapy. However, retinoids also have well-described adverse effects. The quantitative, high-content assay will be useful to screen for less-toxic retinoids or combinations of agents that promote myoblast differentiation in the presence of TGF-β1.     

Dynamics and Binding Modes of Free cdk2 and its Two Complexes with Inhibitors Studied by Computer Simulations

Abstract

This article presents a molecular dynamics (MD) study of the cdk2 enzyme and its two complexes with the inhibitors isopentenyladenine and roscovitine using the Cornell et al. force field from the AMBER software package. The results show that inserting an inhibitor into the enzyme active site does not considerably change enzyme structure but it seemingly changes the distribution of internal motions. The inhibitor causes differences in the domain motions in free cdk2 and in its complexes. It was found out that repulsion of roscovitine N9 substituent causes conformational change on Lys 33 side chain. Isopentenyladenine forms with Lys 33 side chain terminal amino group a hydrogen bond. It implies that the cavity, where N9 substituent of roscovitine is buried, can adopt larger substituent due to Lys 33 side chain flexibility. The composition of electrostatic and van der Waals interactions between the inhibitor and the enzyme were also calculated along both cdk2/inhibitor MD trajectories together with MM-PB/GBSA analysis. These results show that isopentenyladenine-like inhibitors could be more effective after modifications leading to an increase in their van der Waals contact with the enzyme. We suggest that a way leading to better inhibitors occupying isopentenyladenine binding mode could be: to keep N9 and N7 purine positions free, to keep 3,3-dimethylallylamino group at C6 position, and to add, e.g., benzylamino group at C2 position. The results support the idea that the isopentenyladenine binding mode can be used for cdk2 inhibitors design and that all possibilities to improve this binding mode were not uncovered yet.     

The molecular basis of inhibitor resistance in a mammalian mitochondrial cytochrome b mutant

The mitochondrial gene for the cytochrome b of Complex III has been cloned from a mouse L-cell mutant with increased resistance to 2-n-heptyl-4-hydroxyquinoline-N-oxide and other inhibitors which block reactions at the b562 heme group. Nucleotide sequencing revealed that this gene contained a G:A transition on the coding strand at position 14,830. At the amino acid level, this mutation results in the substitution of an aspartic acid residue for a conserved glycine at position 231 of cytochrome b. Based upon current models for the secondary structure of cytochrome b, the altered amino acid lies in close proximity to one of the invariant histidine residues involved in binding the heme groups. Combining this result with the previous biochemical studies of this mutant, we hypothesize that the insertion of this highly charged side chain alters the conformation around the b562 heme group such that 2-n-heptyl-4-hydroxyquinoline-N-oxide and the other inhibitors of this group have reduced access to the inhibitor binding domain.