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Poor glycemic control and dyslipidemia are hallmarks of type 2 diabetes mellitus (T2DM), which predispose to cardiovascular diseases. Peroxisome proliferator-activated receptor-α (

Minor structural modifications of acyclic nucleoside phosphonates can dramatically affect their antiviral properties. This work discloses a shift in the selectivity spectrum of 3-hydroxy-2-(phosphonomethoxy)propyl (HPMP) nucleotides from herpesviruses toward hepatitis B virus (HBV) induced by their acyclic chain 2-substitution with a nonpolar group. Two series of racemic (R,S)-2-methyl-3-hydroxy-2-(phosphonomethoxy)propyl (MHPMP) and (R,S)-2-ethynyl-3-hydroxy-2-(phosphonomethoxy)propyl (EHPMP) nucleotides were initially synthesized. Among these, guanine-containing derivatives exhibited significant anti-HBV activities in the submicromolar range. Enantioenriched MHPMPG and EHPMPG analogues were subsequently obtained by Sharpless asymmetric epoxidation. The (S)-enantiomers possessed an 8- to 26-fold higher potency than the relative (R)-forms. A further comparison of the EC90 values indicated that (S)-EHPMPG inhibited HBV replication more effectively than its 2-methyl analogue. A phosphonodiamidate prodrug of (S)-EHPMPG was thus prepared and found to exert a remarkably high anti-HBV activity (EC50 = 9.27 nM) with excellent selectivity (SI50 > 10,787), proving to be a promising candidate for anti-HBV drug development. ispartof: JOURNAL OF MEDICINAL CHEMISTRY vol:65 issue:13 ispartof: location:United States status: Published online

The main advantage of animal models of infectious diseases over in vitro studies is the gain in the understanding of the complex dynamics between the immune system and the pathogen. While small animal models have practical advantages over large animal models, it is crucial to be aware of their limitations. Although the small animal model at least needs to be susceptible to the pathogen under study to obtain meaningful data, key elements of pathogenesis should also be reflected when compared to humans. Well-designed small animal models for HIV, hepatitis viruses and tuberculosis require, additionally, a thorough understanding of the similarities and differences in the immune responses between humans and small animals and should incorporate that knowledge into the goals of the study. To discuss these considerations, the NIAID hosted a workshop on ‘Small Animal Models for HIV, Hepatitis B, and Tuberculosis’ on May 30, 2019. Highlights of the workshop are outlined below.

Acyclic nucleoside phosphonates incorporating 2,4-diaminotriazine (DAT) as a 5-aza-analog of the 2,4-diamino-pyrimidine (DAPym) nucleobase present in PMEO-DAPyms have been synthesized. The lead PMEO-DAT is as inhibitory against HIV, HBV, MSV and VZV replication as the parent PMEO-DAPym and equally inefficient at markedly affecting replication of HSV-1, HSV-2 and HCMV. A rationale for this similar biological profile is proposed on the basis of structural differences in the active site of the viral DNA polymerases. PMEO-DAT is, however, more selective because, unlike PMEO-DAPym, it does not stimulate secretion of β-chemokines in cultured PBMC., S.d.C. thanks the Spanish MICINN for a post-doctoral Juan de la Cierva contract (JDC-MICINN). We also thank the Spanish MINECO (SAF2012-39760-C02), Comunidad de Madrid (BIPEDD-2-CM S-2010/BMD-2457), and KU Leuven (GOA No. 10/014) for financial support, and Mrs. Lizette van Berckelaer, Mrs. Leen Ingels, Mrs. Leentje Persoons, Mrs. Frieda De Meyer, Mrs. Anita Camps, Mrs. Lies Van den Heurck, Mr. Steven Carmans and Mrs. Sandra Claes for dedicated technical assistance.

Specific sequence changes in codons 70 and 91 of the hepatitis C virus genotype 1b (HCV GT1b) core gene have been associated with increased risk of hepatocellular carcinoma (HCC). Essentially all previous studies were conducted in Asian populations with a wide range of liver disease, and none were conducted specifically in GT1a-infected individuals. We conducted a pilot study in a multiethnic population in the USA with HCV-related cirrhosis to determine if this association extended to GT1a-infected individuals and to determine if other sequence changes in the HCV core gene were associated with HCC risk. HCV core gene sequences from sera of 90 GT1 HCV carriers with cirrhosis (42 with HCC) were analysed using standard RT-PCR-based procedures. Nucleotide sequence data were compared with reference sequences available from GenBank. The frequency of sequence changes in codon 91 was not statistically different between HCC (7/19) and non-HCC (11/22) GT1b carriers. In GT1a carriers, sequence changes in codon 91 were observed less often than in GT1b carriers but were not observed in non-HCC subjects (4/23 vs 0/26, P = 0.03, Fisher's exact test). Sequence changes in codon 70 were not distributed differently between HCC and non-HCC GT1a and 1b carriers. Most importantly, for GT1a carriers, a panel of specific nucleotide changes in other codons was collectively present in all subjects with HCC, but not in any of the non-HCC patients. The utility of this test panel for early detection of HCC in GT1a-infected individuals needs to be assessed in larger populations, including longitudinal studies.

The human norovirus (NoV) polyprotein is cleaved into mature non-structural proteins by both mature NoV protease (Pro, NS6) and its un-cleaved precursor (ProPol, NS6-7). Processing order is well-established with ‘early’ and ‘late’ cleavages, but the governing enzymatic mechanisms are unknown. Enzyme kinetics of a GII Pro and ProPol were analyzed using synthetic peptides representing the five natural polyprotein cleavage sites. The relative efficiency of cleavage of the individual peptides was consistent with established polyprotein processing order, and primarily correlated with enzyme turnover (kcat). Enzymatic efficiencies (kcat/Km) of ProPol at all five sites were equivalent to, or greater than, that of Pro. Binding affinities (Km) for the two least efficiently cleaved sites (p20/VPg, VPg/Pro) were 2–4-fold higher than the other sites. This work further defines the role of ProPol in NoV polyprotein cleavage, and demonstrates that human norovirus polyprotein processing order is primarily an inherent property of enzymatic activity.

A highly active, fluorescence-based, in vitro assay for human Norovirus protease from genogroup I and II viruses was optimized utilizing as little as 0.25 μM enzyme, pH 7.6, and substrate:enzyme of 50–100. Activity in Tris–HCl or sodium phosphate buffers was 2-fold less than HEPES, and 2-fold lower for buffer concentrations over 10 mM. Protease activity at pH 7.6 was 73% (GI) or 63% (GII) of activity at the optimal pH 9.0. Sodium inhibited activity 2–3 fold, while potassium, calcium, magnesium, and manganese inhibited 5–10 fold. Differences in efficiency due to pH, buffer, and cations were due to changes in k cat and not K m . Norovirus protease bound short RNAs representing the 3′ or 5′ ends of the virus, inhibiting protease activity (IC 50 3–5 μM) in a non-competitive manner. Previous reports indicated participation of the protease in the Norovirus replicase complex. The current studies provide initial support for a defined role for the viral protease in Norovirus replication.

L-α-2′-Deoxythreosyl nucleoside phosphonates and their phosphonodiamidate prodrugs with a hypoxanthine, 2,6-diaminopurine, 2-amino-6-cyclopropylaminopurine, 7-deazaadenine, 5-fluorouracil and 5-methylcytosine heterocycle as a nucleobase were synthesized and evaluated for their inhibitory activity against HIV and HBV. The 2,6-diaminopurine modified analogue 23a displayed the most potent activity against HIV, with an EC50 value of 11.17 μM against HIV-1 (IIIB) and an EC50 value of 8.15 μM against HIV-2 (ROD). The application of the prodrug strategy on nucleoside phosphonate 23a led to a 200-fold boost in anti-HIV potency. None of the compounds showed any activity against HBV at the highest concentration tested. ispartof: Organic & Biomolecular Chemistry vol:15 issue:26 pages:5513-5528 ispartof: location:England status: published

The VPg protein of human Norovirus (hNoV) is a multi-functional protein essential for virus replication. The un-cleaved viral precursor protein, ProPol (NS5-6) was 100-fold more efficient in catalyzing VPg nucleotidylylation than the mature polymerase (Pol, NS6), suggesting a specific intracellular role for ProPol. Sequential and single-point alanine substitutions revealed that several positively charged amino acids in the N-terminal region of VPg regulate its nucleotidylylation by ProPol. We provide evidence that VPg directly binds NTPs, inhibition of binding inhibits nucleotidylylation, and NTP binding appears to involve the first 13 amino acids of the protein. Substitution of multiple positively charged amino acids within the first 12 amino acids of the N-terminal region inhibits nucleotidylylation without affecting binding. Substitution of only Lys20 abolishes nucleotidylylation, but not NTP binding. These studies indicate that positively charged amino acids in the first 20 amino acids of hNoV VPg regulate its nucleotidylylation though several potential mechanisms.

We report the activities of a number of thiazolides [2-hydroxyaroyl-N-(thiazol-2-yl)amides] against hepatitis C virus (HCV) genotypes IA and IB, using replicon assays. The structure-activity relationships (SARs) of thiazolides against HCV are less predictable than against hepatitis B virus (HBV), though an electron-withdrawing group at C(5') generally correlates with potency. Among the related salicyloylanilides, the m-fluorophenyl analogue was most promising; niclosamide and close analogues suffered from very low solubility and bioavailability. Nitazoxanide (NTZ) 1 has performed well in clinical trials against HCV. We show here that the 5'-Cl analogue 4 has closely comparable in vitro activity and a good cell safety index. By use of support vector analysis, a quantitative structure-activity relationship (QSAR) model was obtained, showing good predictive models for cell safety. We conclude by updating the mode of action of the thiazolides and explain the candidate selection that has led to compound 4 entering preclinical development.

We report the activities of a number of thiazolides [2-hydroxyaroyl-N-(thiazol-2-yl)amides] against hepatitis C virus (HCV) genotypes IA and IB, using replicon assays. The structure–activity relationships (SARs) of thiazolides against HCV are less predictable than against hepatitis B virus (HBV), though an electron-withdrawing group at C(5′) generally correlates with potency. Among the related salicyloylanilides, the m-fluorophenyl analogue was most promising; niclosamide and close analogues suffered from very low solubility and bioavailability. Nitazoxanide (NTZ) 1 has performed well in clinical trials against HCV. We show here that the 5′-Cl analogue 4 has closely comparable in vitro activity and a good cell safety index. By use of support vector analysis, a quantitative structure–activity relationship (QSAR) model was obtained, showing good predictive models for cell safety. We conclude by updating the mode of action of the thiazolides and explain the candidate selection that has led to compound 4 entering preclinical development.

The acyloxyalkyl derivatives of a model anti-HBV dinucleotide were synthesized and evaluated as orally bioavailable prodrugs. Our studies have led to the identification of the first orally bioavailable dinucleotide prodrugs for further therapeutic development against the hepatitis B virus (HBV).

The relationship between the physical chemistry and biology of self-assembly is poorly understood, but it will be critical to quantitatively understand infection and for the design of antivirals that target virus genesis. Here we take advantage of heteroaryldihydropyrimidines (HAPs), which affect hepatitis B virus (HBV) assembly, to gain insight and correlate in vitro assembly with HBV replication in culture. Based on a low-resolution crystal structure of a capsid-HAP complex, a closely related series of HAPs were designed and synthesized. These differentially strengthen the association between neighboring capsid proteins, alter the kinetics of assembly, and give rise to aberrant structures incompatible with a functional capsid. The chemical nature of the HAP variants correlated well with the structure of the HAP binding pocket. The thermodynamics and kinetics of in vitro assembly had strong and predictable effects on product morphology. However, only the kinetics of in vitro assembly had a strong correlation with inhibition of HBV replication in HepG2.2.15 cells; there was at best a weak correlation between assembly thermodynamics and replication. The correlation between assembly kinetics and virus suppression implies a competition between successful assembly and misassembly, small molecule induced or otherwise. This is a predictive and testable model for the mechanism of action of assembly effectors.

Hepatitis C virus (HCV) is a leading cause of liver cirrhosis and hepatocellular carcinoma worldwide. Currently available treatment options are of limited efficacy, and there is an urgent need for development of alternative therapies. RNA interference (RNAi) is a natural mechanism by which small interfering RNA (siRNA) or short hairpin RNA (shRNA) can mediate degradation of a target RNA molecule in a sequence-specific manner. In this study, we screened in vitro-transcribed 25-bp shRNAs targeting the internal ribosome entry site (IRES) of HCV for the ability to inhibit IRES-driven gene expression in cultured cells. We identified a 44-nt region at the 3! -end of the IRES within which all shRNAs efficiently inhibited expression of an IRES-linked reporter gene. Subsequent scans within this region with 19-bp shRNAs identified even more potent molecules, providing effective inhibition at concentrations of 0.1 nM. Experiments varying features of the shRNA design showed that, for 25-bp shRNAs, neither the size of the loop (4‐10 nt) nor the sequence or pairing status of the ends affects activity, whereas in the case of 19-bp shRNAs, larger loops and the presence of a 3! -UU overhang increase efficacy. A comparison of shRNAs and siRNAs targeting the same sequence revealed that shRNAs were of comparable or greater potency than the corresponding siRNAs. AntiHCV activity was confirmed with HCV subgenomic replicons in a human hepatocyte line. The results indicate that shRNAs, which can be prepared by either transcription or chemical synthesis, may be effective agents for the control of HCV.

Background: The arginine-degrading enzyme, arginine deiminase conjugated to polyethylene glycol (ADI-SS PEG 20 000 mw), reduces extracellular arginine, has minimal toxicity, decreases tumor burden and improves liver function in patients with chronic hepatitis C virus infection (HCV) and inoperable hepatocellular carcinoma (HCC). Reduced extracellular arginine inhibits viral replication through unknown mechanisms. It is hypothesized that ADI-SS PEG 20 000 mw reduces HCV viral titers through nitric oxide (NO)-dependent effects. Methods: The effects of ADI-SS PEG 20 000 mw (dose, 160 IU/m2; three cycles of four once-weekly i.m. injections) on HCV titers, serum NO and plasma arginine, were evaluated using archived plasma from patients with HCC and HCV and in vitro cell model measurements of HCV replication. Results: ADI-SS PEG 20 000 mw selectively inhibited HCV replication in vitro (IC50 = 0.027 IU/mL). Fifteen HCC/HCV patients completed treatment. The HCV titers were reduced by up to 99% in five out of 10 (50%) HCV-serotype 1b patients (P = 0.0093). These patients also experienced significant improvements in liver function (P = 0.0091). There were concomitant reductions of plasma arginine and serum NO levels. The HCV titer was not reduced in HCV-type 2c patients. Conclusion: Reduction of extracellular arginine by ADI-SS PEG 20 000 mw in HCC patients reduces HCV viral titers and improves liver function, possibly through suppression of NO.

Clevudine (CLV) is a nucleoside analog of the unnatural L-configuration that has potent anti-hepatitis B virus (HBV) activity in vitro and in vivo with a favorable toxicity profile in all species tested. In cell culture, CLV is readily phosphorylated to the corresponding 5'-triphosphate form of the compound. The mechanism of action of CLV involves the inhibition of the HBV polymerase by CLV 5'-triphosphate. In vivo efficacy studies performed in the duck and woodchuck models showed marked, rapid inhibition of virus replication and no significant toxicity. In the woodchuck model, there was a dose-dependent delay in viral recrudescence and a reduction or loss of covalently closed circular DNA. In Phase II clinical studies, CLV was well tolerated and exhibited potent antiviral activity at all doses investigated. In Phase III studies in both hepatitis B e antigen (HBeAg)-positive and -negative patients, CLV 30 mg administered once daily demonstrated potent antiviral efficacy and significant biochemical improvement after only 24 weeks of therapy. These effects were sustained in a significant portion of the patients when therapy was stopped after 6 months with no viral rebound occurring in approximately 3 and 16% in HBeAg-positive and -negative patients, respectively. There have been no significant safety or tolerance issues associated with the drug in these studies. Future studies will investigate the safety and tolerance of CLV 30 mg given once daily over 48 weeks and longer.

During the past decade, nucleotide analogs have emerged as novel antiviral agents against hepatitis B virus. Adefovir dipivoxil, a prototype phosphonate analog, has been approved for chronic hepatitis B virus therapy, and additional phosphonate analogs and di- and tri-nucleotides are under development. Several innovative prodrug derivatizations have also been reported to improve the oral bioavailability of nucleotide analogs, which usually carry a negative charge.

In a small controlled study, clevudine, a potent inhibitor of hepadnaviruses, including hepatitis B virus and woodchuck hepatitis virus, suppressed hepatitis delta virus (HDV) viremia in chronically infected woodchucks. Suppression was correlated with the marked reduction of woodchuck hepatitis virus surface antigen in individual animals, consistent with the concept that repression of surface antigen expression may be a useful antiviral strategy for HDV.

Tenofovir disoproxil fumarate (TDF) is a nucleotide analogue approved for treatment of human immunodeficiency virus (HIV) infection. TDF also has been shown in vitro to inhibit replication of wild-type hepatitis B virus (HBV) and lamivudine-resistant HBV mutants and to inhibit lamivudine-resistant HBV in patients and HBV in patients coinfected with the HIV. Data on the in vivo efficacy of TDF against wild-type virus in non-HIV-coinfected or lamivudine-naïve chronic HBV-infected patients are lacking in the published literature. The antiviral effect of oral administration of TDF against chronic woodchuck hepatitis virus (WHV) infection, an established and predictive animal model for antiviral therapy, was evaluated in a placebo-controlled, dose-ranging study (doses, 0.5 to 15.0 mg/kg of body weight/day). Four weeks of once-daily treatment with TDF doses of 0.5, 1.5, or 5.0 mg/kg/day reduced serum WHV viremia significantly (0.2 to 1.5 log reduction from pretreatment level). No effects on the levels of anti-WHV core and anti-WHV surface antibodies in serum or on the concentrations of WHV RNA or WHV antigens in the liver of treated woodchucks were observed. Individual TDF-treated woodchucks demonstrated transient declines in WHV surface antigen serum antigenemia and, characteristically, these woodchucks also had transient declines in serum WHV viremia, intrahepatic WHV replication, and hepatic expression of WHV antigens. No evidence of toxicity was observed in any of the TDF-treated woodchucks. Following drug withdrawal there was prompt recrudescence of WHV viremia to pretreatment levels. It was concluded that oral administration of TDF for 4 weeks was safe and effective in the woodchuck model of chronic HBV infection.

Objectives Investigation of natural ethnopharmacologic extracts exhibiting antiviral potential may lead to the discovery of new therapeutics for the treatment of chronic viral hepatitis infections. Traditional Korean medicinal herbs have been identified that exhibit potency against hepatitis B virus (HBV) and hepatitis C virus (HCV) infections. Research on the antiviral potential of naturally derived extracts is facilitated through the use of appropriate animal and liver cell culture models for these hepatotrophic pathogens. Objectives of this study were to demonstrate antiviral activity of an aqueous extract of herbal formulation KYH-1 in surrogate in vitro assays for HBV and HCV and identify mechanisms of action. Methods Antiviral potency of KYH-1 was measured in tissue culture systems that support replication of the woodchuck hepatitis virus (WHV), and the bovine viral diarrhea virus (BVDV). These assays serve as surrogate models for HBV and HCV, respectively. A recombinant HBV polymerase gene expression assay was used to define a molecular target. Results KYH-1 exhibited potent antiviral activity against WHV and to a lesser extent against BVDV. KYH-1 and its constituent components inhibited HBV polymerase priming in vitro. Additionally, KYH-1 suppressed HBV replication in a human hepatoblastoma cell line. Conclusion Evaluation of naturally derived products for antiviral activity against HBV and HCV in standardized surrogate assays provides a scientific basis for potential use as complementary or alternative medicines. This study provides significant results justifying preclinical evaluation of KYH-1 as an antiviral therapy for HBV infections.

The Eastern woodchuck ( Marmota monax ) harbors a DNA virus (Woodchuck hepatitis virus [WHV]) that is similar in structure and replicative life cycle to the human hepatitis B virus (HBV). Like HBV, WHV infects the liver and can cause acute and chronic hepatitis. Furthermore, chronic WHV infection in woodchucks usually leads to development of hepatocellular carcinoma (HCC) within the first 2-4 years of life. The woodchuck model has been important in the preclinical evaluation of safety and efficacy of the antiviral drugs now in use for treatment of HBV infection and continues to serve as an important, predictive model for innovative forms of therapy of hepatitis B using antiviral nucleosides and immune response modifiers alone or in combination. Almost all woodchucks that become chronic WHV carriers after experimental neonatal inoculation develop HCC with a median HCC-free survival of 24 months and a median life expectancy of 30-32 months. The woodchuck model of viral-induced HCC has been used effectively for the development of new imaging agents for enhancement of detection of hepatic neoplasms by ultrasound and magnetic resonance imaging. The chemoprevention of HCC using long-term antiviral nucleoside therapy has been shown in the woodchuck, and "proof of principal" has been established for some of the innovative, molecular methods for treatment of HCC. The model is available for fundamental investigations of the viral and molecular mechanisms responsible for hepatocarcinogenesis and should have substantial value for future development of innovative methods for chemoprevention and gene therapy of human HCC.

We examined a rational approach to therapy of chronic hepatitis B virus (HBV) infection that utilized the reduction of viral load combined with appropriately timed immune modulation/stimulation. In a placebo-controlled study, chronic woodchuck hepatitis virus (WHV) carrier woodchucks received clevudine (l-FMAU), previously shown to have especially potent and sustained antiviral activity in woodchucks, for 32 weeks followed by WHV surface antigen (WHsAg) alum-adjuvanted vaccine at 32, 36, 40 and 48 weeks. Clevudine induced significant reductions in viraemia, surface antigenaemia, hepatic WHV nucleic acids, and hepatic core and surface antigens. Viral replication markers remained markedly suppressed in 75% of the clevudine-treated woodchucks following drug withdrawal, but remained at high levels in the vaccine monotherapy and placebo groups. Combination drug and vaccine therapy had benefits based on sustained reduction of viraemia, antigenaemia, and hepatic WHV DNA and RNA; inhibition of progression of chronic hepatitis; reduced frequency of chronic liver injury; and delayed onset of hepatocellular carcinoma (HCC). Combination therapy contributed to prevention of HCC in up to 38% of treated carriers, although the growth rate of established HCC was not affected. This study demonstrates enhanced benefits of combination chemo-immunotherapy against viral load and disease progression in chronic hepadnaviral infection, and provides a platform for further development of such treatment regimens.

Integrations of woodchuck hepatitis virus (WHV) DNA and rearrangements of the N-myc 2 gene have been detected frequently in hepatocellular carcinoma (HCC) of Eastern woodchucks (Marmota monax) chronically infected with WHV. Fifty-five hepatocellular neoplasms and matched nontumor hepatic tissue specimens obtained postmortem from 13 chronic WHV carriers were analyzed and the frequency of WHV DNA integrations and of N-myc rearrangements compared in tumors of different size and histologic grade. Four small tumor nodules were classified histologically as adenomas and integrated sequences of WHV DNA were detected in two of the four tumor nodules. In one of the two nodules, there was evidence of N-myc rearrangement. Fifty-one neoplasms were classified as HCC. Seven were grade 1 HCCs. WHV DNA integrations were demonstrated in 43% but none had N-myc rearrangements. Twenty grade 2 HCCs had WHV DNA integrations in 80% and in 38% N-myc rearrangements were present. Twenty-four grade 3 HCCs had integrations of WHV DNA in 79% and N-myc rearrangements in 74%. In two other grade 3 HCCs, rearrangements of N-myc were detected in the absence of WHV DNA integrations. The 12 largest tumors in the series all were grade 2 or 3 HCCs, and in 83%, both WHV DNA integrations and N-myc rearrangements were demonstrated. In conclusion, molecular changes observed in this study suggest a progression of genetic alterations providing either a significant proliferative stimulation and/or a growth advantage in hepatocarcinogenesis of woodchucks with chronic WHV infection.

More than 350 million people worldwide are chronically infected with hepatitis B virus (HBV) (19) and are at high risk of developing chronic hepatitis, hepatic cirrhosis, and hepatocellular carcinoma. Such individuals could benefit immensely from effective therapy, and a combination of antiviral drugs and immunomodulation is a strategy currently under consideration. Successful immunotherapeutic eradication of established HBV infection might be possible if there was better understanding of the underlying mechanisms of chronicity (1, 2, 11, 18, 22, 24, 30, 31, 39; reviewed in references 3 and 26). While approaches using antiviral drugs in combination with alpha interferon (IFN-α) or HBV surface antigen (HBsAg) vaccines have shown promise as therapies for established chronic HBV infection (9, 10, 12, 16, 20, 23, 35), such treatments have not been optimized based on a full understanding of the mechanisms of chronicity. Chronic HBV infection is associated with defects in immunity (reviewed in references 3 and 26) that hinder the development of successful immunotherapy. In contrast to self-limited HBV infection, the HBV carrier appears to be immunologically tolerant. The T helper (Th) cell responses to viral antigens are usually deficient, and antibodies to HBsAg (anti-HBs) are rarely detected. Whether additional B-cell tolerance is involved here is not clear. Antibodies to the HBV core antigen (HBcAg) (anti-HBc antibodies) are detected in carriers with hyporesponsive Th cells to core gene products (HBcAg and HBV e antigen [HBeAg]) because anti-HBc can be elicited via Th-independent mechanisms (32). Th cell responses to HBcAg and HBeAg can be favorable for the carrier when viral replication is diminished during seroconversion to antibodies to HBeAg (anti-HBe) (11, 18, 22, 24, 39). However, T-cell responses in the HBV carrier are generally dysfunctional overall and contribute more to disease progression than to viral clearance (11, 24, 39; reviewed in reference 3). The Eastern woodchuck (Marmota monax) chronically infected with the woodchuck hepatitis virus (WHV) has been used as an animal model to investigate the basic pathogenesis of chronic HBV infection and in the preclinical development of drugs for therapy of HBV (reviewed in references 6, 26, 36, and 38). This animal model mimics many of the immune response features observed in human HBV infections (4, 5, 7, 14, 15, 25, 27-29, 33; reviewed in references 6, 26, 36, and 38) and can predict human responses to antiviral agents as well (21). Experimental studies in chronic WHV carrier woodchucks have been performed using the new and potent antiviral drug 1-(2-fluoro-5-methyl-β-l-arabinofuranosyl)-uracil (l-FMAU) (34), which may represent a promising therapeutic approach for chronic HBV infection. l-FMAU significantly reduces the concentration in serum of both WHV DNA and WHV surface antigen (WHsAg) and also the levels of covalently closed circular viral DNA in the liver (34). A controlled study has now been performed in woodchucks involving treatment with a combination of l-FMAU followed by WHsAg vaccination. The purpose of this report is to show how it is possible using combination therapy to increasingly break immunologic tolerance in chronic WHV carrier woodchucks and to modulate the humoral and cellular immune response profiles toward that observed in self-limited WHV infections.

Novel ring-expanded nucleoside (REN) analogs (1-3) containing 5:7 fused ring systems as the heterocyclic base were found to be potent and selective inhibitors of hepatitis B virus (HBV) replication in cultured human hepatoblastoma 2.2.15 cells. The most active compound, 6-amino-4,5-dihydro-8H-1-(beta-D-ribofuranosyl)imidazo[4,5-e][1,3]diazepine-4,8-dione (1), inhibited the synthesis of intracellular HBV replication intermediates and extracellular virion release in 2.2.15 cells with 50% effective concentration (EC50) of 0.604 and 0.131 microM, respectively. All three compounds had no effect on the synthesis of viral ribonucleic acids (RNA) in 2.2.15 cells. These compounds also exhibited low cellular toxicity in stationary and rapidly growing cell systems.

Noroviruses (NoV) are members of the family Caliciviridae . The human NoV open reading frame 1 (ORF1) encodes a 200-kDa polyprotein which is cleaved by the viral 20-kDa 3C-like protease (Pro, NS6) into 6 nonstructural proteins that are necessary for viral replication. The NoV ORF1 polyprotein is processed in a specific order, with “early” sites (NS1/2-3 and NS3-4) being cleaved rapidly and three “late” sites (NS4-5, NS5-6, and NS6-7) processed subsequently and less efficiently. Previously, we demonstrated that the NoV polyprotein processing order is directly correlated with the efficiency of the enzyme, which is regulated by the primary amino acid sequences surrounding ORF1 cleavage sites. Using fluorescence resonance energy transfer (FRET) peptides representing the NS2-3 and NS6-7 ORF1 cleavage sites, we now demonstrate that the amino acids spanning positions P4 to P2′ (P4-P2′) surrounding each site comprise the core sequence controlling NoV protease enzyme efficiency. Furthermore, the NoV polyprotein self-processing order can be altered by interchanging this core sequence between NS2-3 and any of the three late sites in in vitro transcription-translation assays. We also demonstrate that the nature of the side chain at the P3 position for the NS1/2-3 (Nterm/NTPase) site confers significant influence on enzyme catalysis ( k cat and k cat /K m ), a feature overlooked in previous structural studies. Molecular modeling provides possible explanations for the P3 interactions with NoV protease. IMPORTANCE Noroviruses (NoV) are the prevailing cause of nonbacterial acute gastroenteritis worldwide and pose a significant financial burden on health care systems. Proteolytic processing of the viral nonstructural polyprotein is required for norovirus replication. Previously, the core sequence of amino acids surrounding the scissile bonds responsible for governing the relative processing order had not been determined. Using both FRET-based peptides and full-length NoV polyprotein, we have successfully demonstrated that the core sequences spanning positions P4-P2′ surrounding the NS2-3, NS4-5, NS5-6, and NS6-7 cleavage sites contain all of the structural information necessary to control processing order. We also provide insight into a previously overlooked role for the NS2-3 P3 residue in enzyme efficiency. This article builds upon our previous studies on NoV protease enzymatic activities and polyprotein processing order. Our work provides significant additional insight into understanding viral polyprotein processing and has important implications for improving the design of inhibitors targeting the NoV protease.

A unique series of simple unnatural L-nucleosides that specifically inhibit hepatitis B virus (HBV) replication has been discovered. These molecules have in common a hydroxyl group in the 3'-position (3'-OH) of the beta-L-2'-deoxyribose sugar that confers antiviral activity specifically against hepadnaviruses. Replacement of the 3'-OH broadens activity to other viruses. Substitution in the base decreases antiviral potency and selectivity. Human DNA polymerases and mitochondrial function are not effected. Plasma viremia is reduced up to 8 logs in a woodchuck model of chronic HBV infection. These investigational drugs, used alone or in combination, are expected to offer new therapeutic options for patients with chronic HBV infection.

A unique series of simple “unnatural” nucleosides has been discovered to inhibit hepatitis B virus (HBV) replication. Through structure-activity analysis it was found that the 3′-OH group of the β- l -2′-deoxyribose of the β- l- 2′-deoxynucleoside confers specific antihepadnavirus activity. The unsubstituted nucleosides β- l- 2′-deoxycytidine, β- l -thymidine, and β- l -2′-deoxyadenosine had the most potent, selective, and specific antiviral activity against HBV replication. Human DNA polymerases (α, β, and γ) and mitochondrial function were not affected. In the woodchuck model of chronic HBV infection, viral load was reduced by as much as 10 8 genome equivalents/ml of serum and there was no drug-related toxicity. In addition, the decline in woodchuck hepatitis virus surface antigen paralleled the decrease in viral load. These investigational drugs, used alone or in combination, are expected to offer new therapeutic options for patients with chronic HBV infection.

The octadecyloxyethyl (ODE) and hexadecyloxypropyl (HDP) esters of ( S )-9-[3-hydroxy-2-(phosphonomethoxy)propyl]adenine (HPMPA) are potent inhibitors of orthopoxvirus, herpesvirus, human immunodeficiency virus type 1, and hepatitis B virus replication in vitro. HDP and ODE esters of ( S )-HPMPA and ( R )-HPMPA were evaluated for their activity in hepatitis C virus (HCV) replicon assays using luciferase (1B and 2A replicons) or RNA (1B) quantification. The ODE ester of ( S )-HPMPA [ODE-( S )-HPMPA] was the most active compound, with 50% effective concentrations (EC 50 s) in the 0.69 to 1.31 μM range. HDP and ODE esters of ( R )-HPMPA were severalfold less active, while ( S )-HPMPA and ( R )-HPMPA were inactive. In genotype 1A and 1B replicons analyzed by HCV RNA analysis, ODE-( S )-HPMPA was the most active compound, with EC 50 s of 1.8 and 2.1 μM, respectively.

Cell culture studies in our laboratory previously demonstrated synergistic antiviral activity for the combinations of lamivudine and a novel recombinant hybrid human alpha B/D interferon (rHuαB/D IFN) against hepatitis B virus (HBV) replication. Based on these results, a study was designed to determine if an enhanced antiviral effect with this drug combination could be demonstrated in vivo using the woodchuck hepatitis virus (WHV)/woodchuck experimental model of chronic HBV infection. Both antiviral agents have been shown to be effective against WHV replication in WHV chronic carriers during previous studies by our laboratories. Two combination treatment regimens were compared to matched monotherapies in a placebo-controlled trial. The first used simultaneous administration of rHuαB/D IFN and lamivudine for 24 weeks. The other combination treatment regimen used a staggered dosing schedule of 12 weeks of administration of lamivudine alone, followed by 12 weeks of simultaneous dosing with both drugs, followed by 12 weeks of therapy with rHuαB/D IFN alone. Both treatment regimens with combinations of lamivudine and rHuαB/D IFN were more effective at reducing WHV replication in chronically infected wood-chucks than the corresponding monotherapies. Both combination treatments produced antiviral effects that were at least equal to that expected for additive activity based on estimations generated by Bliss Independence calculations. The staggered treatment regimen reduced viraemia and intrahepatic WHV replication significantly more than that expected for additive interactions, indicating synergistic antiviral effects. These studies demonstrate that combination therapy of chronic WHV infection has enhanced antiviral benefit over corresponding monotherapies and indicate that combination treatment of chronic HBV infection can be superior to therapies using a single antiviral agent.

The synthesis and in vitro anti-hepatitis B virus (HBV) activity of two mononucleoside phosphotriester derivatives of acyclovir incorporating S-acyl-2-thioethyl (SATE) groups are reported. In contrast to the parent nucleoside, the described phosphotriesters emerged as potent and selective inhibitors of HBV replication in HepG2.2.15 cells. This result can be attributed to the unique cellular metabolism of the SATE pronucleotides giving rise to the delivery to acyclovir 5'-monophosphate inside the infected cells. Moreover, the in vitro anti-HBV activities of one of these bis(SATE)phosphotriesters and of (-)-beta-L-2',3'-dideoxy-3'-thiacytidine (lamivudine, 3TC) were compared alone and in combination. Analysis of the combination data indicates that 3TC and the studied SATE pronucleotide of acyclovir exhibited strong synergistic interactions. The present study provides an example where the use of a pronucleotide approach extends the antiviral spectrum of a nucleoside analogue. Given the potency of SATE pronucleotides of acyclovir against HBV in HepG2.2.15 cells, further studies including animal experiments seem warranted to evaluate the potential of these compounds as anti-HBV agents.

Robustaflavone, a naturally occurring biflavanoid isolated from Rhus succedanea , was found to be a potent inhibitor of hepatitis B virus (HBV) replication in 2.2.15 cells, with an effective concentration (EC 50 ) of 0.25 μ M, and a selectivity index (SI, IC 50 /EC 90 ) of 153. Robustaflavone hexaacetate inhibited HBV replication with an EC 50 of 0.73 μ M, but exhibited no cytotoxicity at concentrations up to 1000 μ M. Combinations of robustaflavone with penciclovir and lamivudine displayed synergistic anti-HBV activity, having the most pronounced effects when the combination ratios were similar to the ratio of EC 50 potencies. Thus, a 1:1 combination of robustaflavone and penciclovir exhibited an EC 50 of 0.11 μ M and an SI of 684, while a 10:1 combination of robustaflavone and lamivudine exhibited an EC 50 of 0.054 μ M and an SI of 894. Statistical analyses of the combination data using the Combostat® program confirmed that robustaflavone exhibited synergism with both penciclovir and lamivudine.

Woodchucks were used to study the antiviral activity and toxicity of fialuridine (FIAU; 1,-2'deoxy-2'fluoro-1-beta-D-arabinofuranosyl-5-iodo-uracil). In an initial experiment, groups of six chronic woodchuck hepatitis virus (WHV) carrier woodchucks received daily doses of FIAU by intraperitoneal injection for 4 weeks. At 0.3 mg/kg/d, the antiviral effect was equivocal, but at 1.5 mg/kg/d, FIAU had significant antiviral activity. No evidence of drug toxicity was observed during the 4-week period of treatment or during posttreatment follow-up. In a second experiment, groups of nine WHV carriers or uninfected woodchucks were given 1.5 mg/kg/d of FIAU orally for 12 weeks, and the results compared with placebo-treated controls. After 4 weeks, the serum WHV-DNA concentration in the FIAU-treated carrier group was two to three logs lower than that in the placebo-treated group. After 12 weeks of FIAU treatment, serum WHV DNA was not detectable by conventional dot-blot analysis, hepatic WHV-DNA replicative intermediates (RI) had decreased 100-fold, and hepatic expression of WHV core antigen was remarkably decreased. No evidence of toxicity was observed after 4 weeks, but, after 6 to 7 weeks, food intake decreased and, after 8 weeks, the mean body weights of woodchucks treated with FIAU were significantly lower than controls. Anorexia, weight loss, muscle wasting, and lethargy became progressively severe, and all FIAU-treated woodchucks died or were euthanized 78 to 111 days after treatment began. Hepatic insufficiency (hyperbilirubinemia, decreased serum fibrinogen, elevated prothrombin time), lactic acidosis, and hepatic steatosis were characteristic findings in the final stages of FIAU toxicity in woodchucks. The syndrome of delayed toxicity in woodchucks was similar to that observed previously in humans treated with FIAU, suggesting that the woodchuck should be valuable in future investigations of the molecular mechanisms of FIAU toxicity in vivo and for preclinical toxicological evaluation of other nucleoside analogs before use in patients.

(+)-5′-Noraristeromycin ( 1 ) has shown significant antiviral activity while its 7-deaza analogue 2 is an anti-trypanosomal candidate. To determine the relevance of the 4′-hydroxyl hydrogen in these activities, a derivative of 1 (that is, 3 ) where the C-4′ hydroxyl hydrogen has been replaced by a methyl group has been prepared beginning with palladium (0) mediated coupling of the sodium salt of N 6 -benzoyladenine ( 9 ) and (1 S ,4 R )-4-methoxy-2-cyclopenten-1-yl acetate ( 5 ). The synthesis of compound 5 is described from (1 S ,4 R )-1-[( tert -butyldimethylsilyl)oxy]-4-hydroxycyclopent-2-ene ( 6 ) in three steps. Analogous preparations of the 7-deaza and 8-aza-7-deaza derivatives of 3 related to 2 (that is, 4 and 12 ) are also reported. The new derivatives ( 3 , 4 , and 12 ) failed to show improved antiviral activity. Compound 12 was the only derivative with some anti-trypanosomal activity, giving 40% inhibition of growth at 100 μM against bloodstream forms of a Typanosoma brucei brucei isolate in a standard in vitro screen. This study indicated that the C-4′-hydroxyl hydrogen plays a role in the medicinal properties of 1 and 2 .

(+)-5′-Noraristeromycin has selective activity against hepatitis B virus (HBV) replication in 2.2.15 cells in culture, while the (-) enantiomer was found to be inactive. A modified synthesis is presented for (+)-5′-noraristeromycin.

Robustaflavone, a naturally occurring biflavanoid isolated from the seed kernel extract of Rhus succedanea, was found to be a potent in vitro inhibitor of hepatitis B, with an effective concentration (EC50) of 0.25 μM and an in vitro selectivity index ( IC 50 EC 90 ) of 153. Further studies suggested that inhibition of HBV DNA polymerase is the mechanism of action.

The structure-activity relationship of sixteen 3-deaza, C-4 substituted pyrimidines and imidazo[1,2-c]pyrimidine bases of 1,3-oxathiolanes and 1,3-dioxolanes revealed good anti-HBV activity in 2.2.15 cells transfected with human hepatitis B virus of the imidazo[1,2-c]pyrimidine nucleosides 21, 25 and 29. Two procedures for the preparation of C-4 substituted analogues are reported based on nucleophilic displacement of a sulfonamide or imidazole by a variety of nitrogen nucleophiles.

Noroviruses (NV) are the most common cause of acute gastrointestinal illness in the United States and worldwide. The development of specific antiviral countermeasures has lagged behind that of other viral pathogens, primarily because norovirus disease has been perceived as brief and self-limiting and robust assays suitable for drug discovery have been lacking. The increasing recognition that NV illness can be life-threatening, especially in immunocompromised patients who often require prolonged hospitalization and intensive supportive care, has stimulated new research to develop an effective antiviral therapy. Here, we propose a path forward for evaluating drug therapy in norovirus-infected immunocompromised individuals, a population at high risk for serious and prolonged illness. The clinical and laboratory features of norovirus illness in immunocompromised patients are reviewed, and potential markers of drug efficacy are defined. We discuss the potential design of clinical trials in these patients and how an anti-viral therapy that proves effective in immunocompromised patients might also be used in the setting of acute outbreaks, especially in confined settings such as nursing homes, to block the spread of infection and reduce the severity of illness. We conclude by reviewing the current status of approved and experimental compounds that might be evaluated in a hospital setting.

Various purine β-L-2′,3′-dideoxynucleoside analogues with both sugar and base modifications including β-L-ddG, β-L-ddl, β-L-ddA, 2′-azido-β-L-araddA, 2′-amino-β-L-araddA, 2′,5′-anhydro-β-L-araddA, 2′-azido-β-L-ddA, 2′-amino-β-L-ddA, 2′-fluoro-β-L-ddA, 3′-azido-β-L-ddA, 3′-amino-β-L-ddA, 3′-fluoro-β-L-ddA, 2,6-diamino-β-L-2′,3′-dideoxyfuranosylpurine, 6-cyclopropylamino-β-L-ddA, 2′-azido-6-N-triphenylphosphine-β-L-araddA, 2-amino-6-methylamino-β-L-2′,3′-dideoxyfuranosylpurine, 2-amino-6-cyclopropylamino-β-L-2′,3′-dideoxyfuranosylpurine, 2-amino-6-cyclopentylamino-β-L-2′,3′-dideoxyfuranosylpurine, 2′,3′-didehydro-β-L-ddA and 2′,3′-didehydro-6-N-triphenyl phosphine-β-L-ddA were synthesized and evaluated as potential inhibitors of hepatitis B virus (HBV) replication in HBV DNA-transfected human hepatoblastoma-derived Hep-G2 cells (2.2.15 cells). β-L-ddA, 2′-azido-β-L-ddA, 3′-azido-β-L-ddA, 2″,3′-didehydro-β-L-ddA (β-L-D4A) and a modified base of β-L-D4A, inhibited HBV replication in vitro. β-L-D4A was the more potent and selective antiHBV agent with a 50% effective concentration value of 0.1 μM and a selectivity index of 1800. On the basis of this finding, studies are in progress to synthesize new purine derivatives with the β-L unnatural configuration which hopefully will lead to identifying additional potent and highly selective anti-HBV agents.

ORI-9020, a novel dinucleotide, evaluated in transgenic mice expressing hepatitis B virus (HBV), significantly reduced liver HBV DNA ( P ≤ 0.001). Levels of HBeAg and HBsAg in serum and of HBcAg in liver were not affected by treatment. A minimal effective dosage was determined to be between 1.6 and 0.5 mg/kg of body weight/day, which was similar to that observed for adefovir dipivoxil.

Novel 2'-fluoro-6'-methylene-carbocyclic adenosine (FMCA) monophosphate prodrug (FMCAP) was synthesized and evaluated for its in vitro anti-HBV potency against a lamivudine-entecavir resistant clone (L180M+M204V+S202G). FMCA demonstrated significant antiviral activity against wild-type as well as lamivudine-entecavir resistant triple mutant (L180M+M204V+S202G). The monophosphate prodrug (FMCAP) demonstrated greater than 12-fold (12×) increase in anti-HBV activity without increased cellular toxicity. Mitochondrial and cellular toxicity studies of FMCA indicated that there is no significant toxicity up to 100 μM. Mode of action studies by molecular modeling indicate that the 2'-fluoro moiety by hydrogen bond as well as the Van der Waals interaction of the carbocyclic ring with the phenylalanine moiety of the polymerase promote the positive binding, even in the drug-resistant mutants.

Background Any landmark-based regional anaesthetic technique raises two important issues. The first is the accuracy of placement of the needle and thus the local anaesthetic in a ‘blind' technique and the second is the potential for damage to adjacent structures. We designed a prospective, blinded study in an adult general surgical population to evaluate with ultrasound the placement of the needle tip and local anaesthetic during transversus abdominis plane (TAP) blocks using the landmark-based ‘double-pop' technique. Methods After induction of general anaesthesia, 36 adult patients had a TAP block performed bilaterally using the standard landmark-based technique. Ultrasonography was then used to record the actual needle position and local anaesthetic spread. The anaesthetist performing the block was blinded to the ultrasound images. Results Thirty-six adult patients were included in the study, which was terminated early due to what was considered an unacceptably high level of peritoneal needle placements. The needle tip and local anaesthetic spread were in the correct plane in only 17 (23.6%) of the injections. In the remaining 55 (76.4%), the needle was in the subcutaneous tissue 1 (1.38%), external oblique muscle 1 (1.38%), plane between the external and internal oblique muscles 5 (6.94%), internal oblique muscle 26 (36.1%), transversus abdominis muscle 9 (12.5%), and peritoneum 13 (18%). Conclusions We conclude that the needle and local anaesthetic placement using the standard landmark-based approach to the TAP block is inaccurate, and the incidence of peritoneal placement is unacceptably high.

An efficient method was developed for the synthesis of 6-exocyclic methylene carbocyclic intermediate 4. The Simmons-Smith cyclopropanation protocol was applied on the 6-exocyclic methylene of intermediate 4 and demonstrated its utility for the synthesis of novel class of a spiro-carbocyclic nucleoside analog 8. The titled compound 8 demonstrated a significant antiviral activity against HCV with EC(50) values of 0.273 and 0.368 μM in genotypes 1A and 1B, respectively.

An efficient method was developed for the synthesis of 6-exocyclic methylene carbocyclic intermediate 4. The Simmons-Smith cyclopropanation protocol was applied on the 6-exocyclic methylene of intermediate 4 and demonstrated its utility for the synthesis of novel class of a spiro-carbocyclic nucleoside analog 8. The titled compound 8 demonstrated a significant antiviral activity against HCV with EC(50) values of 0.273 and 0.368 μM in genotypes 1A and 1B, respectively.

Nitazoxanide (NTZ) exhibits potent antiviral activity against hepatitis C virus (HCV) in cell culture. Previously, HCV replicon-containing cell lines resistant to NTZ were selected, but transfer the HCV NTZ-resistance phenotype was not observed following transfection of whole cell RNA. To further explore the nature of the resistance of HCV to NTZ, full length HCV replicon sequences were obtained from two NTZ-resistant (NTZ-11, TIZ-9), and the parental (RP7) cell lines. Numerous nucleotide changes were observed in individual HCV genomes relative to the RP7 HCV consensus sequence, but no common mutations in the HCV non-structural genes or 3'-UTR were detected. A cluster of single nucleotide mutations was found within a 5-base portion of the 5'-UTR in 20/21 HCV replicon sequences from both resistant cell lines. Three mutations (5'-UTR G17A, G18A, C20U) were individually inserted into CON1 ('wild-type') HCV replicons, showed reduced replication (5 to 50-fold), but none conferred resistance to NTZ. RP7, NTZ-11, and TIZ-9 were cured of HCV genomes by serial passage under interferon. Transfection of cured NTZ-11 and TIZ-9 with either whole cell RNAs from RP7, NTZ-11, or TIZ-9, 'wild-type' or the 5'-UTR mutation-containing replicon RNAs exhibited an NTZ-resistance phenotype. TIZ (the active metabolite of NTZ) was found to be inactive against the activity of HCV polymerase, protease, and helicase in enzymatic assays. These data confirm previous speculations that HCV resistance to NTZ is not due to mutations in the virus, and demonstrate that HCV resistance and most likely the antiviral activity of TIZ are due to interactions with cellular target(s).

Promising biological activity in a number of therapeutic areas has been reported for both tricyclic nucleosides and 2'-modified nucleosides. In particular, disubstitution at the C-2' position of nucleosides has resulted in significant activity against the hepatitis C virus (HCV). Combining this with the observation that tricyclic nucleosides developed in our laboratory have been shown to inhibit the RNA-dependent RNA polymerase NS5B led to the design of a series of 2'-modified tricyclic nucleosides. Details of the synthesis, structural characterization, and preliminary biological results are reported.

The in vitro antihepatitis B virus (HBV) activities of eleven novel imidazo[1,2-c]pyrimidin-5(6H)-one dideoxynucleoside analogues in which the sugar ring is 1,3-dioxolane or 1,3-oxathiolane were compared in the chronically HBV-producing human cell line 2.2.15. Seven nucleoside analogues 4, 9, 10, 15, 16, 18, and 24, of which 16 possesses the trans relative stereochemistry, displayed good potency and selectivity towards HBV. The order of decreasing potency at the 90% extracellular DNA inhibition level was 15>16>24≈9>10>18 . None of the tested imidazo[1,2-c]pyrimidines inhibited the replication of HIV-1 in MT-4 cells.