Derivatives of imidazotriazine and pyrrolotriazine C-nucleosides as potential new anti-HCV agents

Previous investigations identified 2′-C-Me-branched ribo-C-nucleoside adenosine analogues, 1, which contains a pyrrolo[2,1-f][1,2,4]triazin-4-amine heterocyclic base, and 2, which contains an imidazo[2,1-f][1,2,4]triazin-4-amine heterocyclic base as two compounds with promising anti-HCV in vitro activity. This Letter describes the synthesis and evaluation of a series of novel analogues of these compounds substituted at the 2-, 7-, and 8-positions of the heterocyclic bases. A number of active new HCV inhibitors were identified but most compounds also demonstrated unacceptable cytotoxicity. However, the 7-fluoro analogue of 1 displayed good potency with a promising cytotherapeutic margin.     

Synthesis of new 2'-beta-C-methyl related triciribine analogues as anti-HCV agents

Ten new beta-D-ribofuranosyl and 2'-beta-C-methyl-beta-D-ribofuranosyl triciribine derivatives 4-13 with various N4 and 6-N substituents on the tricyclic ring were synthesized from the corresponding toyocamycin and new 2'-beta-C-methyl toyocamycin derivatives. The inhibitory studies of these compounds in the HCV replicon assay reveal that some of them possess interesting anti-HCV properties with low cytotoxicity.     

微小RNA-122对丙型肝炎病毒复制及感染治疗影响的研究进展

丙型肝炎病毒(HCV)感染后易演变为慢性肝炎,甚至进展为肝硬化、肝癌。目前尚无有效的预防疫苗,抗病毒药物的疗效也较局限。因此,直接靶向抗病毒且无毒副作用的治疗方法是目前研究的重点。微小RNA(miRNA)是一类小分子非编码RNA,主要通过下调宿主基因表达而发挥生物学功能。miRNA-122(miR-122)在HCV感染中的作用受到关注,探讨其影响HCV复制的具体分子机制对将其作为抗病毒治疗的一个靶目标、研发新型靶向抗HCV治疗药物有重要意义。本文主要就miR-122对HCV复制的影响及其成为潜在治疗靶点的研究现状作一综述。     

7-O-Arylmethylgalangin as a novel scaffold for anti-HCV agents

In spite of potent antiviral activity, suboptimal physicochemical properties of aryl diketo acids (ADKs) necessitates modification of the core 1,3-diketo acid functionality into a novel scaffold. As the metal-binding affinity of the diketo acid is the key to the antiviral activity of ADKs, we anticipated 3,5-dihydroxy-4-oxo arrangement of galangin scaffold would serve as an excellent mimic for the diketo acid functionality. In this study, through synthesis and biological evaluation of various galangin derivatives, we have shown that the diketo acid functionality can be successfully replaced with the galangin scaffold by specific combination of the substituents to result in identification of a novel galangin derivative (3s) with anti-HCV activity (EC₅₀ = 0.9 μM) comparable to the ADK counterpart.     

Current approaches to the search for new restriction endonucleases

The main methodological approaches to the search of new restriction endonucleases are reviewed. These methods include obtaining acellular extracts by ultrasonic desintegration of microbial cells, osmotic shock effects, the effects of organic solvents, mechanical disruption of bacterial cells, biphase division after Albertson and others. The resolving power of any method discussed depends mainly on the level of restriction endonuclease activity, the presence of nonspecific endonucleases in the biomass, the presence of exonucleases and the taxonomy of the used microorganisms.     

Benzohydroxamic acids as potent and selective anti-HCV agents

A diverse collection of 40 derivatives of benzohydroxamic acid (BHAs) of various structural groups were synthesized and tested against hepatitis C virus (HCV) in full-genome replicon assay. Some of these compounds demonstrated an exceptional activity, suppressing viral replication at sub-micromolar concentrations. The compounds were inactive against key viral enzymes NS3, and NS5B in vitro assays, suggesting host cell inhibition target(s). The testing results were consistent with metal coordination by the BHAs hydroxamic group in complex with a target(s). Remarkably, this class of compounds did not suppress poliomyelitis virus (PV) propagation in RD cells indicating a specific antiviral activity of BHAs against HCV.     

Comparative study on the replication of HCV1b genome between wild-type and cell culture-adaptive mutant in regard to sensitivities against anti-HCV drugs

The replicon system, which mimics viral genome replication in culture cells, has been widely used to analyze the genome replication of the hepatitis C virus (HCV). However, most HCV genomes used in the system include adaptive mutations (AMs) that are vital for replication in culture cells despite the nonexistence of such mutations in the genome of wild-type (WT) HCV in patients. In order to study the genome replications of WT HCV, new HCV subgenomic replicon (SGR) systems were established using Huh-7.5-derived cells producing Sec14-like protein 2 constitutively and SGR of KT9 (one of the HCV genotype 1b clones) with WT genome (SGR KT9WT) in this study. The replication efficiency and sensitivities of SGR KT9WT to anti-HCV drugs in the cloned cells permanently bearing replicon RNA, HS55-4 cells, were similar to those of reports using SGR, including AM. The SGR transient transfection system using SGR KT9WT and SGR KT9AM encoding secreted Nano-luciferase and HS55-4C cells established by the elimination of SGR KT9 RNA from HS55-4 cells, however, showed that the replication efficiency of SGR KT9WT was much lower than that of SGR KT9AM under a same condition. Furthermore, the sensitivities of SGR KT9WT to almost all tested anti-HCV reagents, except the inhibitor of miR-122, a cellular factor important for HCV replication, were quite low compared with SGR KT9AM. These results suggested that the new replicon systems might not only provide information about precise responses against new anti-HCV drugs but also reveal novel molecular mechanisms supporting negligent proliferation of HCV.     

Asymmetric synthesis of cyclopropyl-fused 2'-C-methylcarbanucleosides as potential anti-HCV agents

Novel 2'-C-methyl-cyclopropyl-fused carbocyclic nucleosides as potential anti-HCV agents were stereoselectively synthesized, utilizing regioselective cleavage of the isopropylidene group and cyclic sulfate chemistry as key steps.     

Extraneous agents testing for substrates of avian origin and viral vaccines for poultry: Current provisions and proposals for future approaches

In the 1970s the European Pharmacopoeia (Ph. Eur.) established the first requirements for testing starting materials for vaccines and the vaccines themselves. These requirements also cover testing for freedom from extraneous agents of specific pathogen free (SPF) chicken flocks, the embryonated eggs derived from them and viral vaccines for poultry. This was the first common European approach initiated by the Ph. Eur. as an institution of the Council of Europe and it was the beginning of building a scientific basis for vaccine quality. In the following years, the increasingly detailed requirements concerning viral purity also impacted viral vaccines for poultry, SPF chicken flocks and the embryonated eggs derived from them. The core of these requirements is formed by the list of extraneous agents that must be tested for and the accepted test methods. In the early 1990s and in 2004, the next steps were taken towards the harmonisation of quality regulations for the production and testing of veterinary immunological products, this time at the level of the European Community. With the first step, good manufacturing practices (GMP) and good laboratory practices (GLP) were introduced, ensuring more consistent production, validation of production procedures and testing. The next step introduced the risk assessment, which covers the evaluation of the quality of production and control.The intention of these efforts is to contribute to the quality, safety and purity of the products placed on the market. It makes sense that, based on the outcome of the risk-evaluation, a reduction of in-process and final product testing may be called for in certain cases. However, despite the fact that the quality of the starting materials and vaccines has been increased over the years, the provisions of the Ph. Eur. have not been adjusted. Progress made by the manufacturers of starting materials and vaccines with respect to increasing the quality of their products should be recognised. This review gives an analysis of the current provisions of the Ph. Eur. and makes some proposals on how the requirements concerning the testing of extraneous agents could be modified to take into consideration the increase in quality that has been achieved over the past few decades.     

Rate of cirrhosis progression reduced in HIV/HCV co-infected non-responders to anti-HCV therapy

This is a retrospective longitudinal follow-up study of 25 HIV/HCV positive cirrhotic patients not responding to peg-IFN plus ribavirin, and 25 untreated controls matched for age (+/-5 years), gender and Child-Pugh score. The primary endpoint of the study was the incidence of cirrhosis progression (CP) defined as the occurrence of at least one of the following events: death, ascites, jaundice, encephalopathy, gastrointestinal bleeding and hepatocellular carcinoma (HCC). During the median follow-up of 54 months (34-89), four treated (16%) and 13 untreated patients (52%) experienced CP (p = 0.02). Poisson's regression model showed that the independent predictors of CP were Peg-IFN therapy (p = 0.016), positive HIV-RNA (p = 0.024), and altered ALP values (p = 0.012). Peg-IFN therapy seems to slow down the rate of cirrhosis progression also in HIV/HCV co-infected patients nonresponders to anti-HCV therapy, in comparison with untreated patients.     

Synthesis of purine modified 2'-C-methyl nucleosides as potential anti-HCV agents

Based on the anti-hepatitis C activity of 2′-C-methyl-adenosine and 2′-C-methyl-guanosine, a series of new modified purine 2′-C-methyl nucleosides was prepared as potential anti-hepatitis C virus agents. Herein, we report the synthesis of both 6-modified and 2-modified purine 2′-C-methyl-nucleosides along with their anti-HCV replication activity and cytotoxicity in different cells.     

Searching for a new anti-HCV therapy: synthesis and properties of tropolone derivatives

Hepatitis C virus (HCV) is considered one of the most dangerous pathogens since about 3% of the world population is HCV-infected and the virus is a major cause of hepatitis, cirrhosis, and liver carcinoma. A need for a more efficient therapy prompted us to investigate new class of compounds, such as tropolone derivatives that possess antiviral, antibacterial, and antifungal activities. To synthesize bromo- and morpholinomethyl-analogues of tropolone, the previously reported methods were modified. The influence of new derivatives on the activity of the helicase and NTP-ase of HCV was investigated. The most potent inhibitory effect in the fluorometric helicase assay was exerted by 3,7-dibromo-5-morpholinomethyltropolone, for which the IC50 value was at low micromolar range. All the morpholino-derivatives had inhibitory activities higher than those of the non-modified analogues. Low toxicity in a yeast-based toxicity assay indicates that these compounds could be further modified to develop potent inhibitors of the HCV helicase and of viral replication.     

Discovery of pyrimidine nucleoside dual prodrugs and pyrazine nucleosides as novel anti-HCV agents

To explore the application potential of dual prodrug strategies in the development of anti-HCV agents, a variety of sofosbuvir derivatives with modifications at the C4 or N3 position of the uracil moiety were designed and synthesized. Some compounds exhibited potent anti-HCV activities, such as 4e and 8a–8c with similar EC₅₀ values (0.20–0.22?μM) comparative to that of sofosbuvir (EC₅₀?=?0.18?μM) in a genotype 1b based replicon Huh-7 cell line. Moreover, 8b displayed a good human plasma stability profile, and was easily metabolized in human liver microsomes expectantly. On the other hand, aiming to discover novel anti-HCV nucleosides, pyrazin-2(1H)-one nucleosides and their phosphoramidate prodrugs were investigated. Several active compounds were discovered, such as 25e (EC₅₀?=?7.3?μM) and S-29b (EC₅₀?=?19.5?μM). This kind of nucleosides were interesting and would open a new avenue for the development of antiviral agents.     

Current and future approaches using genetically modified mice in endocrine research

Genetically modified mouse models have been used widely to advance our knowledge in the field of endocrinology and metabolism. A number of different approaches to generate genetically modified mice are now available, which provide the power to analyze the role of individual proteins in vivo. However, there are a number of points to be considered in the use and interpretation of these models. This review discusses the advantages and disadvantages involved in the generation and use of different genetically modified mouse models in endocrine research, including conventional techniques (e.g., overexpression, knockout, and knock-in models), tissue- and/or time-specific deletion of target genes [e.g., Cre-loxP and short interfering (si)RNA transgenic approaches], and gene-trap approaches to undertake functional genomics. This review also highlights the many factors that should be considered when assessing the phenotype of these mouse models, many of which are relevant to all murine physiological studies. These approaches are a powerful means by which to dissect the function of genes and are revolutionizing our understanding of endocrine physiology and metabolism.     

Identifying new targets in leukemogenesis using computational approaches

There is a need to identify novel targets in Acute Lymphoblastic Leukemia (ALL), a hematopoietic cancer affecting children, to improve our understanding of disease biology and that can be used for developing new therapeutics. Hence, the aim of our study was to find new genes as targets using in silico studies; for this we retrieved the top 10% overexpressed genes from Oncomine public domain microarray expression database; 530 overexpressed genes were short-listed from Oncomine database. Then, using prioritization tools such as ENDEAVOUR, DIR and TOPPGene online tools, we found fifty-four genes common to the three prioritization tools which formed our candidate leukemogenic genes for this study. As per the protocol we selected thirty training genes from PubMed. The prioritized and training genes were then used to construct STRING functional association network, which was further analyzed using cytoHubba hub analysis tool to investigate new genes which could form drug targets in leukemia. Analysis of the STRING protein network built from these prioritized and training genes led to identification of two hub genes, SMAD2 and CDK9, which were not implicated in leukemogenesis earlier. Filtering out from several hundred genes in the network we also found MEN1, HDAC1 and LCK genes, which re-emphasized the important role of these genes in leukemogenesis. This is the first report on these five additional signature genes in leukemogenesis. We propose these as new targets for developing novel therapeutics and also as biomarkers in leukemogenesis, which could be important for prognosis and diagnosis.     

Current and future management approaches for rheumatoid arthritis

With the introduction of new disease-modifying antirheumatic drugs (DMARDs) and other therapeutic agents, the management of rheumatoid arthritis (RA) has shifted toward earlier, more aggressive therapy. The ultimate goal is to prevent structural joint damage that leads to pain and functional disability. Early diagnosis of RA is therefore essential, and early DMARD treatment combined with nonsteroidal anti-inflammatory drugs is recommended. Combination DMARD regimens and new biologic agents (anti-tumor necrosis factor [TNF] therapies [infliximab, etanercept] and the interleukin [IL]-1 antagonist [anakinra]) have emerged as viable options for early treatment of RA patients. These new biologic agents and future nonbiologic agents that target proteins in signaling cascades are likely to change the landscape of RA treatments.     

Novel rank-based approaches for discovery and replication in genome-wide association studies

In recent years, genome-wide association studies (GWAS) have uncovered a large number of susceptibility variants. Nevertheless, GWAS findings provide only tentative evidence of association, and replication studies are required to establish their validity. Due to this uncertainty, researchers often focus on top-ranking SNPs, instead of considering strict significance thresholds to guide replication efforts. The number of SNPs for replication is often determined ad hoc. We show how the rank-based approach can be used for sample size allocation in GWAS as well as for deciding on a number of SNPs for replication. The basis of this approach is the "ranking probability": chances that at least j true associations will rank among top u SNPs, when SNPs are sorted by P-value. By employing simple but accurate approximations for ranking probabilities, we accommodate linkage disequilibrium (LD) and evaluate consequences of ignoring LD. Further, we relate ranking probabilities to the proportion of false discoveries among top u SNPs. A study-specific proportion can be estimated from P-values, and its expected value can be predicted for study design applications.     

Effect of hepatitis C virus (HCV) NS5B-nucleolin interaction on HCV replication with HCV subgenomic replicon

We previously reported that nucleolin, a representative nucleolar marker, interacts with nonstructural protein 5B (NS5B) of hepatitis C virus (HCV) through two independent regions of NS5B, amino acids 208 to 214 and 500 to 506. We also showed that truncated nucleolin that harbors the NS5B-binding region inhibited the RNA-dependent RNA polymerase activity of NS5B in vitro, suggesting that nucleolin may be involved in HCV replication. To address this question, we focused on NS5B amino acids 208 to 214. We constructed one alanine-substituted clustered mutant (CM) replicon, in which all the amino acids in this region were changed to alanine, as well as seven different point mutant (PM) replicons, each of which harbored an alanine substitution at one of the amino acids in the region. After transfection into Huh7 cells, the CM replicon and the PM replicon containing NS5B W208A could not replicate, whereas the remaining PM replicons were able to replicate. In vivo immunoprecipitation also showed that the W208 residue of NS5B was essential for its interaction with nucleolin, strongly suggesting that this interaction is essential for HCV replication. To gain further insight into the role of nucleolin in HCV replication, we utilized the small interfering RNA (siRNA) technique to investigate the knockdown effect of nucleolin on HCV replication. Cotransfection of replicon RNA and nucleolin siRNA into Huh7 cells moderately inhibited HCV replication, although suppression of nucleolin did not affect cell proliferation. Taken together, our findings strongly suggest that nucleolin is a host component that interacts with HCV NS5B and is indispensable for HCV replication.