Polymeric black tea polyphenols modulate the localization and activity of 12-O-tetradecanoylphorbol-13-acetate-mediated kinases in mouse skin: Mechanisms of their anti-tumor-promoting action

Polymeric black tea polyphenols (PBPs) have been shown to possess anti-tumor-promoting effects in two-stage skin carcinogenesis. However, their mechanisms of action are not fully elucidated. In this study, mechanisms of PBP-mediated antipromoting effects were investigated in a mouse model employing the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). Compared to controls, a single topical application of TPA to mouse skin increased the translocation of protein kinase C (PKC) from cytosol to membrane. Pretreatment with PBPs 1-3 decreased TPA-induced translocation of PKC isozymes (α, β, η, γ, ε) from cytosol to membrane, whereas PBPs 4 and 5 were less effective. The levels of PKCs δ and ζ in cytosol/membrane were similar in all the treatment groups. Complementary confocal microscopic evaluation showed a decrease in TPA-induced PKCα fluorescence in PBP-3-pretreated membranes, whereas pretreatment with PBP-5 did not show a similar decrease. Based on the experiments with specific enzyme inhibitors and phosphospecific antibodies, both PBP-3 and PBP-5 were observed to decrease TPA-induced level and/or activity of phosphatidylinositol 3-kinase (PI3K) and AKT1 (pS473). An additional ability of PBP-3 to inhibit site-specific phosphorylation of PKCα at all three positions responsible for its activation [PKCα (pT497), PKC PAN (βII pS660), PKCα/βII (pT638/641)] and AKT1 at the Thr308 position, along with a decrease in TPA-induced PDK1 protein level, correlated with the inhibition of translocation of PKC, which may impart relatively stronger chemoprotective activity to PBP-3 than to PBP-5. Altogether, PBP-mediated decrease in TPA-induced PKC phosphorylation correlated well with decreased TPA-induced NF-κB phosphorylation and downstream target proteins associated with proliferation, apoptosis, and inflammation in mouse skin. Results suggest that the antipromoting effects of PBPs are due to modulation of TPA-induced PI3K-mediated signal transduction.     

2-O-β-d-Glucopyranosyl-sn-glycerol based analogues of sulfoquinovosyldiacylglycerols (SQDG) and their role in inhibiting Epstein-Barr virus early antigen activation

New sulfoquinovosyldiacylglycerols derived from 2-O-β-d-glucopyranosyl-sn-glycerol, carrying acyl chains of various length on the glycerol moiety, were prepared through a convenient synthetic procedure in which a sulfonate is introduced at the C-6 position of glucose by oxidation of a thioacetate in the presence of the unprotected secondary hydroxyl groups, and tested for their anti-tumor-promoting activity using a short-term in vitro assay for Epstein-Barr virus early antigen (EBV-EA) activation. Our study has allowed to ascertain the role of the 6′-sulfonate group and the need of a free hydroxyl group on the glycerol moiety in inhibiting the EBV activation promoted by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA).     

Inhibitors of HIV-1 attachment. Part 2: An initial survey of indole substitution patterns

The effects of introducing simple halogen, alkyl, and alkoxy substituents to the 4, 5, 6 and 7 positions of 1-(4-benzoylpiperazin-1-yl)-2-(1H-indol-3-yl)ethane-1,2-dione, an inhibitor of the interaction between HIV gp120 and host cell CD4 receptors, on activity in an HIV entry assay was examined. Small substituents at C-4 generally resulted in increased potency whilst substitution at C-7 was readily tolerated and uniformly produced more potent HIV entry inhibitors. Substituents deployed at C-6 and, particularly, C-5 generally produced a modest to marked weakening of potency compared to the prototype. Small alkyl substituents at N-1 exerted minimal effect on activity whilst increasing the size of the alkyl moiety led to progressively reduced inhibitory properties. These studies establish a basic understanding of the indole element of the HIV attachment inhibitor pharmacophore.     

Identification of Hepatitis C Virus NS5A Inhibitors

Using a cell-based replicon screen, we identified a class of compounds with a thiazolidinone core structure as inhibitors of hepatitis C virus (HCV) replication. The concentration of one such compound, BMS-824, that resulted in a 50% inhibition of HCV replicon replication was ∼5 nM, with a therapeutic index of >10,000. The compound showed good specificity for HCV, as it was not active against several other RNA and DNA viruses. Replicon cells resistant to BMS-824 were isolated, and mutations were identified. A combination of amino acid substitutions of leucine to valine at residue 31 (L31V) and glutamine to leucine at residue 54 (Q54L) in NS5A conferred resistance to this chemotype, as did a single substitution of tyrosine to histidine at amino acid 93 (Y93H) in NS5A. To further explore the region(s) of NS5A involved in inhibitor sensitivity, genotype-specific NS5A inhibitors were used to evaluate a series of genotype 1a/1b hybrid replicons. Our results showed that, consistent with resistance mapping, the inhibitor sensitivity domain also mapped to the N terminus of NS5A, but it could be distinguished from the key resistance sites. In addition, we demonstrated that NS5A inhibitors, as well as an active-site inhibitor that specifically binds NS3 protease, could block the hyperphosphorylation of NS5A, which is believed to play an essential role in the viral life cycle. Clinical proof of concept has recently been achieved with derivatives of these NS5A inhibitors, indicating that small molecules targeting a nontraditional viral protein like NS5A, without any known enzymatic activity, can also have profound antiviral effects on HCV-infected subjects.Hepatitis C virus (HCV) is the major causative agent for non-A, non-B hepatitis worldwide, which affects more than 3% of the world population. HCV establishes chronic infections in a large percentage of infected individuals, increasing the risk for developing liver cirrhosis and, in some cases, hepatocellular carcinoma. Although the current standard of care for HCV infection involves the use of PEGylated interferon and ribavirin, a large proportion of patients fail to respond to this therapy, and treatment is associated with frequent and sometimes serious side effects (9). Given the limited efficacy of the current therapy, the development of safer and more effective therapies is of tremendous importance.HCV is a positive-strand RNA virus belonging to the family Flaviviridae. The HCV genome consists of a ∼9.6-kb RNA with a large open reading frame encoding a polyprotein of ∼3,010 amino acids. The polyprotein is cleaved co- and posttranslationally by both cellular and viral proteases into at least 10 different products (10, 11). The viral proteins required for RNA replication include NS3, NS4A, NS4B, NS5A, and NS5B (4, 19). NS3 consists of an amino-terminal protease domain required for the cleavage of the remaining nonstructural proteins and a carboxyl-terminal helicase/NTPase domain (8, 11, 30). NS4A serves as a cofactor for NS3 protease and helicase activities (8). NS4B is an integral membrane protein involved in the formation of the membranous web, where HCV replication complexes are thought to assemble (7). NS5A is a membrane-associated phosphoprotein present in basally phosphorylated (p56) and hyperphosphorylated (p58) forms (15, 31). It was previously reported that only p58-defective mutants could be complemented in trans (1), and NS5A is involved in HCV virion production (22, 34), suggesting that different forms of NS5A exert multiple functions at various stages of the viral life cycle. The N terminus of NS5A (domain I) has been crystallized in alternative dimer forms and contains zinc- and RNA-binding domains (20, 33). The ability of NS5A to bind to zinc (32) and RNA (14) has been demonstrated in vitro. NS5A has been shown to interact with a number of host proteins, is implicated in interferon resistance in vivo, and has been the subject of several reviews (13, 21). NS5B functions as the viral RNA-dependent RNA polymerase (2). Previous studies have shown that the NS3-NS5B proteins are all essential for HCV replication and are believed to form the HCV replicase complex (4, 18, 19).The development of the cell-based HCV replicon system provides a means for the large-scale screening of HCV inhibitors against multiple viral targets. The use of a cell-based replication assay likely includes essential functions that previously could not be evaluated with in vitro enzyme assays. The disadvantages for the advancement of HCV inhibitors targeting nonenzymatic proteins are (i) the potential for structure-activity relationships (SAR) to be difficult to interpret based on the complexity of cell-based systems, (ii) the lack of a system for validation, and (iii) difficulty in predicting if in vitro potency can translate into in vivo effect. Therefore, during the process of developing HCV NS5A inhibitors, we established a series of assays and checkpoints prior to entering the clinic. This is the first report in a series of articles detailing the development of HCV NS5A inhibitors that has culminated in the demonstration of clinical efficacy for this novel mechanistic class of HCV inhibitor (25).In this report, we have used a previously described cell-based approach (26) to identify a novel compound that specifically inhibits HCV RNA replication. Through the use of resistance selection, we have demonstrated that the inhibitor targets the HCV NS5A protein, thereby establishing that the function of NS5A in replication can be inhibited by small molecules. In addition, using genotype-specific inhibitors, we have further shown that the N terminus of NS5A plays an essential role in compound activity by both 50% effective concentration (EC₅₀) determinations as well as a functional assay to evaluate NS5A hyperphosphorylation.     

Reduction of diffusion barriers in isolated rat islets improves survival, but not insulin secretion or transplantation outcome

For people with type 1 diabetes and severe hypoglycemic unawareness, islet transplants offer hope for improving the quality of life. However, islet cell death occurs quickly during or after transplantation, requiring large quantities of islets per transplant. The purpose of this study was to determine whether poor function demonstrated in large islets was a result of diffusion barriers and if removing those barriers could improve function and transplantation outcomes. Islets were isolated from male DA rats and measured for cell viability, islet survival, glucose diffusion and insulin secretion. Modeling of diffusion barriers was completed using dynamic partial differential equations for a sphere. Core cell death occurred in 100% of the large islets (diameter >150 μm), resulting in poor survival within 7 days after isolation. In contrast, small islets (diameter <100 μm) exhibited good survival rates in culture (91%). Glucose diffusion into islets was tracked with 2-NBDG; 4.2 μm/min in small islets and 2.8 μm/min in large islets. 2-NBDG never permeated to the core cells of islets larger than 150 μm diameter. Reducing the diffusion barrier in large islets improved their immediate and long-term viability in culture. However, reduction of the diffusion barrier in large islets failed to improve their inferior in vitro insulin secretion compared to small islets, and did not return glucose control to diabetic animals following transplantation. Thus, diffusion barriers lead to low viability and poor survival for large islets, but are not solely responsible for the inferior insulin secretion or poor transplantation outcomes of large versus small islets.     

pC6-2/caspase-6 system to purify glutathione-S-transferase-free recombinant fusion proteins expressed in Escherichia coli

Glutathione-S-transferase (GST) fusion protein expression vectors are often employed for the expression and purification of proteins in Escherichia coli. GST is then removed by site-specific proteolysis using thrombin. However, the presence of internal thrombin cleavage sites in expressed proteins can severely affect the purification of intact proteins. Cysteine-dependent aspartate-specific proteases (caspases) are efficient enzymes with defined substrate specificity. Unlike most of the proteases used for the removal of affinity tags, caspases do not leave any amino acids at the amino-terminus of cleaved proteins. We have engineered the caspase-6 site VEMD in a pGEX vector to give the pC6-2 vector. The caspase-6 can be easily removed after cleavage. Here, we describe the detailed protocol for purifying proteins using our pC6-2/caspase-6 expression and purification system. The cleavage by caspase-6 occurs in <30 min and the entire procedure can be completed in 2 d.     

pH-Dependent Changes in Photoaffinity Labeling Patterns of the H1 Influenza Virus Hemagglutinin by Using an Inhibitor of Viral Fusion

The hemagglutinin (HA) protein undergoes a low-pH-induced conformational change in the acidic milieu of the endosome, resulting in fusion of viral and cellular membranes. A class of compounds that specifically interact with the HA protein of H1 and H2 subtype viruses and inhibit this conformational change was recently described (G. X. Luo et al., Virology 226:66–76, 1996, and J. Virol. 71:4062–4070, 1997). In this study, purified HA trimers (bromelain-cleaved HA [BHA]) are used to examine the properties and binding characteristics of these inhibitors. Compounds were able to inhibit the low-pH-induced change of isolated trimers, as detected by resistance to digestion with trypsin. Protection from digestion was extremely stable, as BHA-inhibitor complexes could be incubated for 24 h in low pH with almost no change in BHA structure. One inhibitor was prepared as a radiolabeled photoaffinity analog and used to probe for specific drug interactions with the HA protein. Analysis of BHA after photoaffinity analog binding and UV cross-linking revealed that the HA2 subunit of the HA was specifically radiolabeled. Cross-linking of the photoaffinity analog to BHA under neutral (native) pH conditions identified a stretch of amino acids within the α-helix of HA2 that interact with the inhibitor. Interestingly, cross-linking of the analog under acidic conditions identified a different region within the HA2 N terminus which interacts with the photoaffinity compound. These attachment sites help to delineate a potential binding pocket and suggest a model whereby the BHA is able to undergo a partial, reversible structural change in the presence of inhibitor compound.Influenza virus contains a lipid envelope that must fuse with host cell membranes in order to initiate virus infection (42, 43, 49). The hemagglutinin (HA) protein, a trimeric glycoprotein embedded in the viral membrane, is responsible for specific binding to cell surface sialic acid-containing receptors (46) and for the fusion of the two membranes (51). Although the mechanism of viral fusion is not fully elucidated, it is known that the fusion event is preceded by a conformational change occurring in the HA trimer that is triggered by the decreasing pH encountered during endosomal passage of the virus (23, 43, 49, 50). The HA trimer is composed of three identical monomers, each containing two protein subunits (designated HA1 and HA2) attached to each other via a disulfide linkage (36, 52). These monomer subunits are formed from a single chain precursor HA (HA0) that undergoes cleavage during transport from the Golgi to the cell surface (27). Entry of the influenza virus into host cells is facilitated through receptor binding by the HA1 subunit to the sialic acid-containing receptor. The conformational change brought on by the low pH of the endosome exposes the hydrophobic amino terminus of the HA2 subunit, which is believed to be a trigger in the fusion process (8, 17, 19, 40). It is postulated that the native state of the HA is a spring-loaded coiled coil and upon acidification, the hydrophobic fusion peptide is translocated toward the target membrane (9–11). This exposed hydrophobic amino terminus is believed to mediate fusion with the cell membrane (8, 19).Influenza virus HA can be cleaved from viral membrane surfaces with bromelain protease to create a soluble form of the protein (bromelain-cleaved HA [BHA]) (5, 52). The soluble HA remains a trimer with properties identical to those of the native membrane bound protein (44). Upon acidification, BHA undergoes a conformational change and forms rosettes caused by the aggregation of the exposed hydrophobic fusogenic domains of the HA2 subunit (14, 40). In this conformation, the BHA is susceptible to trypsin digestion, while it is resistant to this protease in its native conformation (15, 40).We have previously reported on the identification of a class of compounds that can inhibit influenza virus fusion (29, 30). These compounds are able to inhibit the low pH induced conformational change in the HA protein of H1 and H2 subtype viruses but not of the H3 subtype virus. Of these three subtypes, precise structural information is available only for H3 HA (8, 20, 37, 38, 45, 48). Previously a model of H1 HA was constructed using H3 HA crystal structure data (52) and a potential fusion inhibitor-binding pocket was identified within HA2 based on resistant mutation analysis and inhibitor selectivity (30). In order to probe this binding model and better understand the mechanism of action of these compounds, experiments were carried out with isolated H1 BHA. Various analogs were able to protect BHA from protease digestion following acid treatment and subsequent neutralization. A radiolabeled analog which possessed a photoactivatable azide moiety was synthesized (16). Affinity labeling at a neutral or acidic pH produced very different profiles of labeled amino acids, although in each case the amino acids were in or near the proposed binding pocket in the HA2. The consequences of the differences in HA2 photoaffinity labeling patterns with regard to the mechanism of action of these fusion inhibitors are discussed below.