Tylophorine Analogs Allosterically Regulates Heat Shock Cognate Protein 70 And Inhibits Hepatitis C Virus Replication.

Tylophorine analogs have been shown to exhibit diverse activities against cancer, inflammation, arthritis, and lupus in vivo. In this study, we demonstrated that two tylophorine analogs, DCB-3503 and rac-cryptopleurine, exhibit potent inhibitory activity against hepatitis C virus (HCV) replication in genotype 1b Con 1 isolate. The inhibition of HCV replication is at least partially mediated through cellular heat shock cognate protein 70 (Hsc70). Hsc70 associates with the HCV replication complex by primarily binding to the poly U/UC motifs in HCV RNA. The interaction of DCB-3503 and rac-cryptopleurine with Hsc70 promotes the ATP hydrolysis activity of Hsc70 in the presence of the 3' poly U/UC motif of HCV RNA. Regulating the ATPase activity of Hsc70 may be one of the mechanisms by which tylophorine analogs inhibit HCV replication. This study demonstrates the novel anti-HCV activity of tylophorine analogs. Our results also highlight the importance of Hsc70 in HCV replication.

Determinants of individual variation in intracellular accumulation of anti-HIV nucleoside analog metabolites.

Individual variation in response to antiretroviral therapy is well-known, but it is not clear if demographic characteristics such as gender, age, and ethnicity are responsible for the variation. To optimize anti-HIV therapy and guide antiretroviral drug discovery, determinants that cause variable responses to therapy need to be evaluated. We investigated the determinants of intracellular concentrations of nucleoside analogs using peripheral blood mononuclear cells from 40 healthy donors. We observed individual differences in the concentrations of the intracellular nucleoside analogs; the mean concentrations of the triphosphate metabolite of ethynylstavudine (4'-Ed4T), zidovudine (AZT), and lamivudine (3TC) were 0.71 pmol/10(6) cells (minimum and maximum, 0.10 and 3.00 pmol/10(6) cells, respectively), 0.88 pmol/10(6) cells (minimum and maximum, 0.10 and 15.18 pmol/10(6) cells, respectively), and 1.70 pmol/10(6) cells (minimum and maximum, 0.20 and 7.73 pmol/10(6) cells, respectively). Gender and ethnicity had no effect on the concentration of 4'-Ed4T and 3TC metabolites. There was a trend for moderation of the concentrations of AZT metabolites by gender (P = 0.17 for gender·metabolite concentration). We observed variability in the activity and expression of cellular kinases. There was no statistically significant correlation between thymidine kinase 1 (TK-1) activity or expression and thymidine analog metabolite concentrations. The correlation between the activity of deoxycytidine kinase (dCK) and the 3TC monophosphate metabolite concentration showed a trend toward significance (P = 0.1). We observed an inverse correlation between the multidrug-resistant protein 2 (MRP2) expression index and the concentrations of AZT monophosphate, AZT triphosphate, and total AZT metabolites. Our findings suggest that the observed variation in clinical response to nucleoside analogs may be due partly to the individual differences in the intracellular concentrations, which in turn may be affected by the cellular kinases involved in the phosphorylation pathway and ATP-binding cassette (ABC) transport proteins.

A novel class of meso-tetrakis-porphyrin derivatives exhibits potent activities against hepatitis C virus genotype 1b replicons in vitro.

Recent years have seen the rapid advancement of new therapeutic agents against hepatitis C virus (HCV) in response to the need for treatment that is unmet by interferon (IFN)-based therapies. Most antiviral drugs discovered to date are small molecules that modulate viral enzyme activities. In the search for highly selective protein-binding molecules capable of disrupting the viral life cycle, we have identified a class of anionic tetraphenylporphyrins as potent and specific inhibitors of the HCV replicons. Based on the structure-activity relationship studies reported herein, meso-tetrakis-(3,5-dicarboxy-4,4'-biphenyl) porphyrin was found to be the most potent inhibitor of HCV genotype 1b (Con1) replicon systems but was less effective against the genotype 2a (JFH-1) replicon. This compound induced a reduction of viral RNA and protein levels when acting in the low nanomolar range. Moreover, the compound could suppress replicon rebound in drug-treated cells and exhibited additive to synergistic effects when combined with protease inhibitor BILN 2061 or with IFN-alpha-2a. Our results demonstrate the potential use of tetracarboxyphenylporphyrins as potent anti-HCV agents.

Impact of novel human immunodeficiency virus type 1 reverse transcriptase mutations P119S and T165A on 4'-ethynylthymidine analog resistance profile.

2',3'-Didehydro-3'-deoxy-4'-ethynylthymidine (4'-Ed4T), a derivative of stavudine (d4T), has potent activity against human immunodeficiency virus and is much less inhibitory to mitochondrial DNA synthesis and cell growth than its progenitor, d4T. 4'-Ed4T triphosphate was a better reverse transcriptase (RT) inhibitor than d4T triphosphate, due to the additional binding of the 4'-ethynyl group at a presumed hydrophobic pocket in the RT active site. Previous in vitro selection for 4'-Ed4T-resistant viral strains revealed M184V and P119S/T165A/M184V mutations on days 26 and 81, respectively; M184V and P119S/T165A/M184V conferred 3- and 130-fold resistance to 4'-Ed4T, respectively. We investigated the relative contributions of these mutations, engineered into the strain NL4-3 background, to drug resistance, RT activity, and viral growth. Viral variants with single RT mutations (P119S or T165A) did not show resistance to 4'-Ed4T; however, M184V and P119S/T165A/M184V conferred three- and fivefold resistance, respectively, compared with that of the wild-type virus. The P119S/M184V and T165A/M184V variants showed about fourfold resistance to 4'-Ed4T. The differences in the growth kinetics of the variants were not more than threefold. The purified RT of mutants with the P119S/M184V and T165A/M184V mutations were inhibited by 4'-Ed4TTP with 8- to 13-fold less efficiency than wild-type RT. M184V may be the primary resistance-associated mutation of 4'-Ed4T, and P119S and T165A are secondary mutations. On the basis of our findings and the results of structural modeling, a virus with a high degree of resistance to 4'-Ed4T (e.g., more than 50-fold resistance) will be difficult to develop. The previously observed 130-fold resistance of the virus with P119S/T165A/M184V to 4'-Ed4T may be partly due to mutations both in the RT sequence and outside the RT sequence.

Comparative study of the persistence of anti-HIV activity of deoxynucleoside HIV reverse transcriptase inhibitors after removal from culture.

BACKGROUND: Most in vitro assays of drug potency may not adequately predict the performance in vivo. Methods to assess the persistence of antiviral activity of deoxynucleoside analogs, which require intracellular activation to the active metabolites that can persist in cells, will be important for designing dosages, combination regimens, and assessing treatment compliance. Using an HIV-IIIB/TZM-bl indicator cell culture system, we assessed the ability of an inhibitor to protect cells from infection and to delay viral rebound after removal of inhibitor from culture. RESULTS: The order of protection of cells from HIV-infection was 4'-Ed4T > LFD4C > DDI > D4T > 3TC > AZT > FTC > NVP. The fold-increase in EC50 to delay viral rebound was DDI < 4'-Ed4T < LFD4C < FTC < D4T < 3TC < NVP < AZT. The ranking of persistence of anti-HIV activity of the inhibitors based on the two-component assay was DDI > 4'-Ed4T > LFD4C > FTC = D4T > 3TC > NVP > AZT. CONCLUSION: The persistence ranking was derived from assays based on measures of single viral replication-cycle and cumulative inhibition at multiple time-points. Therefore, a better indicator of the pharmacodynamic property of an inhibitor. The persistence of anti-HIV activity assay may complement in vitro potency assays to better predict in vivo performance of nucleoside analogs.

Intracellular metabolism and persistence of the anti-human immunodeficiency virus activity of 2',3'-didehydro-3'-deoxy-4'-ethynylthymidine, a novel thymidine analog.

The therapeutic benefits of current antiretroviral therapy are limited by the evolution of drug-resistant virus and long-term toxicity. Novel antiretroviral compounds with activity against drug-resistant viruses are needed. 2',3'-didehydro-3'-deoxy-4'-ethynylthymidine (4'-Ed4T), a novel thymidine analog, has potent anti-human immunodeficiency virus (HIV) activity, maintains considerable activity against multidrug-resistant HIV strains, and is less inhibitory to mitochondrial DNA synthesis in cell culture than its progenitor stavudine (D4T). We investigated the intracellular metabolism and anti-HIV activity of 4'-Ed4T. The profile of 4'-Ed4T metabolites was qualitatively similar to that for zidovudine (AZT), with the monophosphate metabolite as the major metabolite, in contrast to that for D4T, with relatively poor formation of total metabolites. The first phosphorylation step for 4'-Ed4T in cells was more efficient than that for D4T but less than that for AZT. The amount of 4'-Ed4T triphosphate (4'-Ed4TTP) was higher than that of AZTTP at 24 h in culture. There was a dose-dependent accumulation of 4'-Ed4T diphosphate and 4'-Ed4TTP on up-regulation of thymidylate kinase and 3-phosphoglycerate kinase expression in Tet-On RKO cells, respectively. The anti-HIV activity of 4'-Ed4T in cells persisted even after 48 h of drug removal from culture in comparison with AZT, D4T, and nevirapine (NVP). The order of increasing persistence of anti-HIV activity of these compounds after drug removal was 4'-Ed4T > D4T > AZT > NVP. In conclusion, with the persistence of 4'-Ed4TTP and persistent anti-HIV activity in cells, we anticipate less frequent dosing and fewer patient compliance issues than for D4T. 4'-Ed4T is a promising antiviral candidate for HIV type 1 chemotherapy.

Structure-activity studies of phenanthroindolizidine alkaloids as potential antitumor agents.

Five phenanthroindolizidine alkaloids (PA) were chemically synthesized and seven were isolated from Tylophora atrofolliculata. To facilitate future drug design of phenanthroindolizidine alkaloids as potential antitumor agents, we have explored the structure-activity relationships (SAR) of this class of compounds. We demonstrated that DCB-3503 and tylophorinidine (PA-7) were among the most active compounds against tumor growth both in vitro and in vivo. In the hepatocellular carcinoma cell line HepG2, the GI(50)s of DCB-3503 and PA-7 were 35+/-5 nM and 11+/-5 nM, respectively. DCB-3503 and PA-7 significantly inhibited HepG2 tumor growth in nude mice at a dose of 9 mg/kg given by intraperitoneal (ip) injections twice a day every third day for a total of four cycles (P<0.05 for DCB-3503 and P<0.01 for PA-7). Their potent antitumor activities correlated with their potent NF-kappaB-inhibitory effects and their cyclin D1 down-regulatory effects.

Eriocalyxin B inhibits nuclear factor-kappaB activation by interfering with the binding of both p65 and p50 to the response element in a noncompetitive manner.

Nuclear factor-kappaB (NF-kappaB) has been recognized to play a critical role in cell survival and inflammatory processes. It has become a target for intense drug development for the treatment of cancer, inflammatory, and autoimmune diseases. Here, we describe a potent NF-kappaB inhibitor, eriocalyxin B (Eri-B), an ent-kauranoid isolated from Isodon eriocalyx, an anti-inflammatory remedy. The presence of two alpha,beta-unsaturated ketones give this compound the uniqueness among the ent-kauranoids tested. Eri-B inhibited the NF-kappaB transcriptional activity but not that of cAMP response element-binding protein. It suppressed the transcription of NF-kappaB downstream gene products including cyclooxygenase-2 and inducible nitric-oxide synthase induced by tumor necrosis factor-alpha or lipopolysaccharide in macrophages and hepatocarcinoma cells. Chromatin immunoprecipitation assay indicated that Eri-B selectively blocked the binding between NF-kappaB and the response elements in vivo without affecting the nuclear translocation of the transcription factor. Down-regulation of the endogenous p65 protein sensitized the cells toward the action of the compound. Furthermore, in vitro binding assays suggested that Eri-B reversibly interfered with the binding of p65 and p50 subunits to the DNA in a noncompetitive manner. In summary, this study reveals the novel action of a potent NF-kappaB inhibitor that could be potentially used for the treatment of a variety of NF-kappaB-associated diseases. Modification of the structure of this class of compounds becomes the key to the control of the behavior of the compound against different cellular signaling pathways.

Novel mechanism of inhibition of nuclear factor-kappa B DNA-binding activity by diterpenoids isolated from Isodon rubescens.

The development of specific inhibitors that can block nuclear factor-kappaB (NF-kappaB) activation is an approach for the treatment of cancer, autoimmune, and inflammatory diseases. Several diterpenoids, oridonin, ponicidin, xindongnin A, and xindongnin B were isolated from the herb Isodon rubescens. These compounds were found to be potent inhibitors of NF-kappaB transcription activity and the expression of its downstream targets, cyclooxygenase-2 and inducible nitric-oxide synthase. The mechanisms of action of the diterpenoids against NF-kappaB are similar, but significant differences were also identified. All of the diterpenoids directly interfere with the DNA-binding activity of NF-kappaB to its response DNA sequence. Oridonin and ponicidin have an additional impact on the translocation of NF-kappaB from the cytoplasm to nuclei without affecting IkappaB-alpha phosphorylation and degradation. The effect of these compounds on the interaction of NF-kappaB with consensus DNA sequences is unique. Different inhibitory effects were observed when NF-kappaB bound to various DNA sequences. Both p65/p65 and p50/p50 homodimers, as well as p65/p50 heterodimer association with their responsive DNA, were inhibited. Kinetic studies on NF-kappaB-DNA interaction indicate that the diterpenoids decrease the B(max app) but have no effect on K(d app). This suggests that this class of compounds interacts with both p65 and p50 subunits at a site other than the DNA binding site and subsequently modulates the binding affinity of the transcription factor toward DNA with different NF-kappaB binding sequences. The diterpenoid structure could therefore serve as a scaffold for the development of more potent and selective NF-kappaB inhibitors that target regulated gene transcription.

Novel 4'-substituted stavudine analog with improved anti-human immunodeficiency virus activity and decreased cytotoxicity.

The antiviral drug 2',3'-didehydro-3'-deoxythymidine (D4T; also know as stavudine and Zerit), which is used against human immunodeficiency virus (HIV), causes delayed toxicity (peripheral neuropathy) in long-term use. After examining a series of 2',3'-didehydro-3'-deoxy-4'-substituted thymidine (4'-substituted D4T) analogs, 4'-ethynyl D4T was found to have a fivefold-better antiviral effect and to cause less cellular and mitochondrial toxicity than D4T. The antiviral activity of this compound can be reversed by dThd but not by dCyd. The compound acted synergistically with beta-L-2',3'-deoxy-3'-thiacytidine (also known as lamivudine) and beta-L-2',3'-dideoxy-2',3'-didehydro-5-fluorocytidine (also known as elvucitabine) and additively with 2',3'-dideoxyinosine (also known as didanosine and Videx) and 3'-azido-3'-deoxythymidine (also known as Retovir and zidovudine) against HIV. 4'-Ethynyl D4T is phosphorylated by purified human thymidine kinase 1 (TK-1) from CEM cells with a faster relative V(max) and a lower K(m) value than D4T. The efficiency of TK-1 in the phosphorylation of 4'-ethynyl D4T is fourfold better than that of D4T. While D4T is broken down by the catabolic enzyme thymidine phosphorylase, the level of breakdown of 4'-ethynyl D4T was below detection. Since 4'-ethynyl D4T has increased anti-HIV activity and decreased toxicity and interacts favorably with other currently used anti-HIV drugs, it should be considered for further development as an anti-HIV drug.

Novel role of 3-phosphoglycerate kinase, a glycolytic enzyme, in the activation of L-nucleoside analogs, a new class of anticancer and antiviral agents.

l-Nucleoside analogs are a new class of clinically active antiviral and anticancer agents. The phosphorylation of these analogs from diphosphate to triphosphate metabolites is crucial for their biological action. We studied the role of 3-phosphoglycerate kinase, a glycolytic enzyme, in the metabolism of l-nucleoside analogs, using small interfering RNAs to down-regulate the amount of this enzyme in HelaS3 and 2.2.15 cells, chosen as models for studying the impact of the enzyme on the anticancer and antihepatitis B virus activities of these analogs. Decrease in the expression of 3-phosphoglycerate kinase led to a corresponding decrease in the formation of the triphosphate metabolites of l-nucleoside analogs (but not d-nucleoside analogs), resulting in detrimental effects on their activity. The enzyme is important for generating as well as maintaining the steady state levels of l-nucleotides in the cells, thereby playing a key role in the activity of l-nucleoside analogs against human immunodeficiency virus, hepatitis B virus, and cancer. This study also indicates a structure-based distinction in the metabolism of l- and d-nucleoside analogs, disputing the classic notion that nucleoside diphosphate kinases are responsible for the phosphorylation of all classes of nucleoside analog diphosphates.