Brent Korba

Small-Molecule Effectors of Hepatitis B Virus Capsid Assembly Give Insight into Virus Life Cycle

ABSTRACT 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.

Treatment of norovirus infections: Moving antivirals from the bench to the bedside

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 antiviral 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.

Immunogenic effects of woodchuck hepatitis virus surface antigen vaccine in combination with antiviral therapy: breaking of humoral and cellular immune tolerance in chronic woodchuck hepatitis virus infection.

Objective: A rational treatment strategy for chronic hepatitis B virus (HBV) infection might involve the modulation of immunity after the reduction of viremia and antigenemia. This strategy was tested in woodchucks chronically infected with the woodchuck hepatitis virus (WHV) by combining antiviral treatment with 1-(2-fluoro-5-methyl-β-L-arabinofuranosyl)-uracil (L-FMAU) and therapeutic vaccination with WHV surface antigen (WHsAg). Methods: Chronic WHV carriers were treated with L-FMAU or placebo for 32 weeks. Half the woodchucks in each group then received four injections of a conventional WHsAg vaccine during the next 16 weeks. Results: Vaccination alone elicited low-level antibody to WHsAg (anti-WHs) in most carriers but did not affect serum WHV DNA, WHsAg or liver enzyme responses. Carriers treated first with L-FMAU to reduce WHV DNA and WHsAg and then vaccinated developed similar low-level anti-WHs and normalized liver enzymes. Following vaccinations, WHsAg-specific cell-mediated immunity (CMI) was demonstrated in both groups, but was significantly enhanced in carriers treated with L-FMAU, and was broadened to include WHV core antigen (WHcAg) and selected peptide epitopes of WHcAg and WHsAg. Anti-WHs and associated CMI to WHcAg and WHsAg were observed after drug discontinuation in half of the carriers that received L-FMAU alone. Conclusions: Vaccination with WHsAg following treatment with L-FMAU disrupted virus-specific humoral and cell-mediated immune tolerance in chronic WHV infection and enhanced the immune response profiles beyond those seen with monotherapies alone. The combination therapy resulted in immune response profiles that resembled those observed during resolution of WHV infection. The results in woodchucks demonstrate the feasibility of using such a combination therapy for the control of chronic HBV infection in humans.

Enhanced antiviral benefit of combination therapy with lamivudine and famciclovir against WHV replication in chronic WHV carrier woodchucks

Cell culture studies in our laboratory and others have previously demonstrated synergistic antiviral activity for combinations of 3TC (lamivudine) and penciclovir against Hepatitis B Virus (HBV) replication and the Duck Hepatitis B Virus (DHBV). Based on these results, a study was designed to determine if an enhanced antiviral effect with combinations of 3TC and famciclovir (FCV, oral prodrug of penciclovir) 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 in previous studies by our laboratories. The antiviral effects of four different combinations of lamivudine and FCV were found to be greater than those observed for the corresponding monotherapies. All four combination treatments produced antiviral effects that were at least equal to that expected for additive activity based on estimations generated by Bliss Independence calculations. Two of the combination treatments produced antiviral effects that were significantly greater than that expected for additive effects, indicative of synergistic antiviral interactions. 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.

Simvastatin potentiates the anti-hepatitis B virus activity of FDA-approved nucleoside analogue inhibitors in vitro.

Statins are 3-hydroxyl-3-methylglutaryl coenzyme A (HMG CoA) reductase inhibitors used for the treatment of hypercholesterolemia. We report that a particular statin, simvastatin (SIM), exhibits strong in vitro anti-HBV activity. Moreover, a combination of SIM with each of the individual nucleos(t)ide analogues lamivudine (LMV), adefovir (ADV), tenofovir (TEN) and entecavir (ETV), showed synergistic antiviral activity. Combination drug treatments were performed in the HepG2.2.15 cell line. Compound combinations were centered on a mixture designed to deliver approximately equipotent (not necessarily equimolar) concentrations of each agent, based on the ninety percent viral inhibition monotherapy values. SIM interacted favorably with all four licensed anti-HBV nucleos(t)ide analogues, especially at molar ratios that approximate combinations likely to be used clinically. As the relative concentration of SIM was raised to an excess, the overall favorability of the interactions progressively increased. SIM displayed about equal degrees of synergy with ADV and TDF. The highest degree of synergy was observed at the 300:1 combination of SIM with ETV. Interactions with LMV were the least favorable. The in vitro potential shown here may greatly augment anti-HBV therapy clinically.

Identification of HBV DNA sequences that are predictive of response to lamivudine therapy

Numerous studies have shown that resistance to long‐term lamivudine therapy occurs in as many as ⅔ of hepatitis B virus (HBV) chronic carriers. Additional studies have shown that reversion of HBV mutations in the precore/core promoter region conferring an HBeAg‐negative phenotype/genotype can occur in up to 30% of lamivudine‐treated patients. In this study, sequences of the HBV polymerase and precore/core coding regions in 26 HBV‐infected patients (24 with HBeAg‐negative virus infection, 25 genotype D, 1 genotype A) treated for 27 to 53 months with lamivudine were analyzed to determine the relationship between pretreatment HBV DNA sequence patterns and long‐term treatment response, and the effect of therapy on the status of HBV precore mutations. Reversions of precore mutations A1762T/G1764A and G1896A were observed in 29% and 25% of patients, respectively, but none became HBeAg‐positive. These data are consistent with previously published reversion frequencies for 2 other groups of lamivudine‐treated patients. Two naturally‐occurring DNA polymorphisms at aa91 and aa256 of the HBV polymerase were observed in the pretreatment serum samples, which correlated with extended treatment failure. In conclusion, reversion of mutations conferring an HBeAg‐negative phenotype occur relatively frequently under lamivudine therapy. Furthermore, at least in HBeAg‐negative patients infected predominately with HBV genotype D, specific viral DNA sequences which are present before therapy appear to be useful as predictors of long‐term response to lamivudine treatment. (HEPATOLOGY 2004;39:64–73.)

Robustaflavone, a potential non-nucleoside anti-hepatitis B agent

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 (EC50) of 0.25 microM, and a selectivity index (SI, IC50/EC90) of 153. Robustaflavone hexaacetate inhibited HBV replication with an EC50 of 0.73 microM, but exhibited no cytotoxicity at concentrations up to 1000 microM. 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 EC50 potencies. Thus, a 1:1 combination of robustaflavone and penciclovir exhibited an EC50 of 0.11 microM and an SI of 684, while a 10:1 combination of robustaflavone and lamivudine exhibited an EC50 of 0.054 microM 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.

Clevudine: a potent inhibitor of hepatitis B virus in vitro and in vivo

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.

Mutations in the conserved woodchuck hepatitis virus polymerase FLLA and YMDD regions conferring resistance to lamivudine

During more than 104 weeks of treatment with lamivudine (3TC) in chronic woodchuck hepatitis virus (WHV) carrier woodchucks, viral recrudescence occurred. Analysis of WHV DNA polymerase from woodchuck serum samples by PCR followed by DNA sequencing demonstrated that all samples were wild type at the conserved YMDD motif in domain C. Four of the six 3TC-treated woodchucks showed a mixture of the wild-type Ala (GCT) and the mutant Thr (ACT) at the conserved amino acid residue 566 (FLLA) in domain B of the WHV polymerase region. The appearance of the A566T mutation was temporally associated with viral recrudescence. This change is analogous with the amino acid 181 (FLLA) in HBV where 3TC selects for a change from Ala to Thr in humans. In the woodchuck, the Ala to Thr change in the polymerase gene results in a mutation of the WHV surface protein (amino acid 377) from Trp (TGG) to an opal codon (TGA), which may prematurely terminates the polypeptide. Three WHV molecular infectious clones were constructed to study this mutation in greater detail in vitro: A566T, analogous to A181T in HBV; M589V, analogous to the M204V in HBV; and the double mutant A566T/M589V, analogous to A181T/M204V in HBV. These mutants exhibited drug-sensitivity and replication profiles that paralleled those reported for analogous HBV variants. In transfected Huh7 cells, WHV containing the M589V mutation conferred at least 100-fold increased resistance to 3TC, but replicated approximately 5-fold less efficiently than wild-type virus as judged by both extracellular virus production and intracellular DNA replicative forms. In contrast, A566T mutant was approximately 10-fold more resistant to 3TC, replicated intracellularly as well as wild type, but produced 10-fold lower levels of virions than wild type. These findings are consistent with the observation that the A566T mutation alters the overlapping WHV surface antigen reading frame. WHV carrying mutations in the conserved YMDD motif, while not directly selected during lamivudine therapy in WHV carrier woodchucks, are replication competent in cell culture indicating the potential for their emergence in treated animals. These results further illustrate the utility of the WHV/woodchuck model to studies of HBV-drug resistance.

Parallel solid-phase synthesis of nucleoside phosphoramidate libraries.

Combinatorial chemistry is playing an increasingly prominent role in the process of drug discovery. A nucleic acid-based (NAB) scaffold can be engineered to create functional group and topological diversity in a library. Described herein is the parallel solid-phase synthesis of combinatorial libraries of nucleoside phosphoramidates, and the first evaluation of antiviral activity against hepatitis B virus (HBV).