Class A capsid assembly modulator apoptotic elimination of hepatocytes with high HBV core antigen level in vivo is dependent on de novo core protein translation.

Capsid assembly is critical in the hepatitis B virus (HBV) life cycle, mediated by the viral core protein. Capsid assembly is the target for new anti-viral therapeutics known as capsid assembly modulators (CAMs) of which the CAM-aberrant (CAM-A) class induces aberrant shaped core protein structures and leads to hepatocyte cell death. This study aimed to identify the mechanism of action of CAM-A modulators leading to HBV-infected hepatocyte elimination where CAM-A-mediated hepatitis B surface antigen (HBsAg) reduction was evaluated in a stable HBV replicating cell line and in AAV-HBV-transduced C57BL/6, C57BL/6 SCID, and HBV-infected chimeric mice with humanized livers. Results showed that in vivo treatment with CAM-A modulators induced pronounced reductions in hepatitis B e antigen (HBeAg) and HBsAg, associated with a transient alanine amino transferase (ALT) increase. Both HBsAg and HBeAg reductions and ALT increase were delayed in C57BL/6 SCID and chimeric mice, suggesting that adaptive immune responses may indirectly contribute. However, CD8+ T cell depletion in transduced wild-type mice did not impact antigen reduction, indicating that CD8+ T cell responses are not essential. Transient ALT elevation in AAV-HBV-transduced mice coincided with a transient increase in endoplasmic reticulum stress and apoptosis markers, followed by detection of a proliferation marker. Microarray data revealed antigen presentation pathway (major histocompatibility complex class I molecules) upregulation, overlapping with the apoptosis. Combination treatment with HBV-specific siRNA demonstrated that CAM-A-mediated HBsAg reduction is dependent on de novo core protein translation. To conclude, CAM-A treatment eradicates HBV-infected hepatocytes with high core protein levels through the induction of apoptosis, which can be a promising approach as part of a regimen to achieve functional cure. IMPORTANCE: Treatment with hepatitis B virus (HBV) capsid assembly modulators that induce the formation of aberrant HBV core protein structures (CAM-A) leads to programmed cell death, apoptosis, of HBV-infected hepatocytes and subsequent reduction of HBV antigens, which differentiates CAM-A from other CAMs. The effect is dependent on the de novo synthesis and high levels of core protein.

Feasibility, indications, and radiographically confirmed diagnoses of standing extremity cone beam computed tomography in the horse.

OBJECTIVE: To report on the feasibility, indications, and diagnostic yield of cone beam computed tomography (CBCT) of horses' extremities performed under standing sedation. STUDY DESIGN: Retrospective clinical case series. SAMPLE POPULATION: Fifty-nine CBCT examinations in 58 horses. METHODS: Examinations were categorized for indications for CBCT dependent on a suspicion, presence, or absence of a diagnosis prior to CBCT. The number of acquisitions per examination, total time for the examination, diagnostic score of each acquisition (diagnostic, diagnostic-compromised, nondiagnostic), and additional diagnostic information regarding preexisting diagnostic information were recorded. RESULTS: Three (median) acquisitions were performed per examination in a median study time of 14 minutes. In 24 of 33 cases with a suspected diagnosis, this diagnosis was confirmed or definitively refuted; in seven of 33 cases, the suspected diagnosis was refuted without a new diagnosis; and, in two of 33 cases, the suspected diagnosis could not be confirmed nor could a new diagnosis be made. In five of nine cases without a preexisting diagnosis, a diagnosis was established. In 16 cases with a diagnosis prior to CBCT, additional information was recorded, or a surgical plan was prepared. In 14 of 18 cases in which additional contrast techniques were used, additional information was gained. CONCLUSION: Standing CBCT of the horses' extremities is feasible and can produce diagnostic information in a timely fashion. CLINICAL SIGNIFICANCE: The results provide evidence of the practicality and diagnostic potential of standing CBCT of horses' extremities.

Antiviral Properties and Mechanism of Action Studies of the Hepatitis B Virus Capsid Assembly Modulator JNJ-56136379.

Capsid assembly is a critical step in the hepatitis B virus (HBV) life cycle, mediated by the core protein. Core is a potential target for new antiviral therapies, the capsid assembly modulators (CAMs). JNJ-56136379 (JNJ-6379) is a novel and potent CAM currently in phase II trials. We evaluated the mechanisms of action (MOAs) and antiviral properties of JNJ-6379 in vitro Size exclusion chromatography and electron microscopy studies demonstrated that JNJ-6379 induced the formation of morphologically intact viral capsids devoid of genomic material (primary MOA). JNJ-6379 accelerated the rate and extent of HBV capsid assembly in vitro JNJ-6379 specifically and potently inhibited HBV replication; its median 50% effective concentration (EC50) was 54 nM (HepG2.117 cells). In HBV-infected primary human hepatocytes (PHHs), JNJ-6379, when added with the viral inoculum, dose-dependently reduced extracellular HBV DNA levels (median EC50 of 93 nM) and prevented covalently closed circular DNA (cccDNA) formation, leading to a dose-dependent reduction of intracellular HBV RNA levels (median EC50 of 876 nM) and reduced antigen levels (secondary MOA). Adding JNJ-6379 to PHHs 4 or 5 days postinfection reduced extracellular HBV DNA and did not prevent cccDNA formation. Time-of-addition PHH studies revealed that JNJ-6379 most likely interfered with postentry processes. Collectively, these data demonstrate that JNJ-6379 has dual MOAs in the early and late steps of the HBV life cycle, which is different from the MOA of nucleos(t)ide analogues. JNJ-6379 is in development for chronic hepatitis B treatment and may translate into higher HBV functional cure rates.

Effect of Plasma Protein Binding on the Anti-Hepatitis B Virus Activity and Pharmacokinetic Properties of NVR 3-778.

High plasma protein binding (PPB) levels not only affect drug-target engagement but can also impact exposure of hepatocytes to antivirals and thereby affect antiviral activity. In this study, we assessed the effect of PPB on the antiviral activity of NVR 3-778, a sulfamoylbenzamide capsid assembly modulator (CAM). To this end, primary human hepatocyte (PHH) medium was spiked with plasma proteins. First, the effect of plasma proteins on the hepatitis B virus (HBV) infection assay was evaluated. The addition of plasma proteins neither decreased cell viability nor affected HBV DNA secretion or intracellular HBV RNA accumulation. In contrast, the secretion and intracellular amount of HBV proteins were induced with increasing amounts of plasma proteins. Next, the antiviral activity of NVR 3-778 was demonstrated by multiple assays while PPB and the time-dependent disappearance of the parent drug were quantified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Plasma proteins strongly decreased the free fraction of NVR 3-778, resulting in a physiologically relevant in vitro hepatocyte exposure. NVR 3-778 displayed a high PPB level, while the antiviral activity was reduced approximately only 4-fold. The disconnect between the high PPB level and the only moderate shift of the antiviral activity was explained by the rapid hepatic clearance of NVR 3-778 in the absence of plasma proteins. This study highlights the use of PHHs as a model to accurately determine the antiviral activity by capturing PPB, clearance, and liver distribution. It is advantageous to consider both pharmacokinetics and pharmacodynamics for selection of HBV antiviral drug candidates and for successful extrapolation of in vitro data to clinical studies.

Novel Potent Capsid Assembly Modulators Regulate Multiple Steps of the Hepatitis B Virus Life Cycle.

The assembly of hepatitis B virus (HBV) core protein (HBc) into capsids represents a critical step of viral replication. HBc has multiple functions during the HBV life cycle, which makes it an attractive target for antiviral therapies. Capsid assembly modulators (CAMs) induce the formation of empty capsid or aberrant capsid devoid of pregenomic RNA (pgRNA) and finally block relaxed circular DNA neosynthesis and virion progeny. In this study, the novel CAMs JNJ-827 and JNJ-890 were found to be potent inhibitors of HBV replication with respective half-maximal effective concentrations of 4.7 and 66 nM, respectively, in HepG2.117 cells. Antiviral profiling in differentiated HepaRG (dHepaRG) cells and primary human hepatocytes revealed that these compounds efficiently inhibited HBV replication, as well as de novo establishment of covalently closed circular DNA (cccDNA). In addition to these two known effects of CAMs, we observed for the first time that a CAM, here JNJ-827, when added postinfection for a short-term period, significantly reduced hepatitis B e antigen (HBeAg) secretion without affecting the levels of cccDNA amount, transcription, and hepatitis B surface antigen (HBsAg) secretion. This inhibitory activity resulted from a direct effect of JNJ-827 on HBeAg biogenesis. In a long-term treatment condition using persistently infected dHepaRG cells, JNJ-827 and JNJ-890 reduced HBsAg concomitantly with a decrease in viral total RNA and pgRNA levels. Altogether, these data demonstrate that some CAMs could interfere with multiple functions of HBc in the viral life cycle.

Design and synthesis of tetrahydropyridopyrimidine based Toll-Like Receptor (TLR) 7/8 dual agonists.

In a continuing effort to discover novel TLR agonists, herein we report on the discovery and structure-activity relationship of novel tetrahydropyridopyrimidine TLR 7/8 agonists. Optimization of this series towards dual agonist activity and a high clearance profile resulted in the identification of compound 52a1. Evaluation in vivo revealed an interferon stimulated response (ISG) in mice with limited systemic exposure and demonstrated the potential in antiviral treatment or as a vaccine adjuvant.

Discovery of selective 2,4-diaminoquinazoline toll-like receptor 7 (TLR 7) agonists.

The discovery of a novel series of highly potent quinazoline TLR 7/8 agonists is described. The synthesis and structure-activity relationship is presented. Structural requirements and optimization of this series toward TLR 7 selectivity afforded the potent agonist 48. Pharmacokinetic and pharmacodynamic studies highlighted 48 as an orally available endogenous interferon (IFN-α) inducer in mice.

Capsid Assembly Modulators Have a Dual Mechanism of Action in Primary Human Hepatocytes Infected with Hepatitis B Virus.

Hepatitis B virus (HBV) capsid assembly is a critical step in the propagation of the virus and is mediated by the core protein. Due to its multiple functions in the viral life cycle, core became an attractive target for new antiviral therapies. Capsid assembly modulators (CAMs) accelerate the kinetics of capsid assembly and prevent encapsidation of the polymerase-pregenomic RNA (Pol-pgRNA) complex, thereby blocking viral replication. CAM JNJ-632 is a novel and potent inhibitor of HBV replication in vitro across genotypes A to D. It induces the formation of morphologically intact viral capsids, as demonstrated by size exclusion chromatography and electron microscopy studies. Antiviral profiling in primary human hepatocytes revealed that CAMs prevented formation of covalently closed circular DNA in a dose-dependent fashion when the compound was added together with the viral inoculum, whereas nucleos(t)ide analogues (NAs) did not. This protective effect translated into a dose-dependent reduction of intracellular HBV RNA levels as well as reduced HBe/cAg and HBsAg levels in the cell culture supernatant. The same observation was made with another CAM (BAY41-4109), suggesting that mechanistic rather than compound-specific effects play a role. Our data show that CAMs have a dual mechanism of action, inhibiting early and late steps of the viral life cycle. These effects clearly differentiate CAMs from NAs and may translate into higher functional cure rates in a clinical setting when given alone or in combination with the current standard of care.

Synthesis and evaluation of novel HCV replication inhibitors.

Direct acting antiviral agents to cure hepatitis C virus (HCV) infection has emerged as the gold standard therapy. Along with protease inhibitors, nucleoside polymerase inhibitors and non-nucleoside polymerase inhibitors, the inhibition of NS5a has proved to be an effective way to treat HCV patients. Here we report on novel HCV NS5a inhibitors which were synthesized and evaluated in the HCV replicon assay. A series of inhibitors were formed by a cycloaddition reaction in parallel to establish new leads and explore the effects of unsymmetrical cap substitution. This led to the identification of several triazoles with picomolar potency in vitro against hepatitis C virus.

RADIOGRAPHIC MEASUREMENTS OF HOOF BALANCE ARE SIGNIFICANTLY INFLUENCED BY A HORSE'S STANCE.

Hoof balance radiographs are commonly used as the basis for corrective farriery decision-making in horses, however there are limited published data quantifying effects of the stance of the horse or the horizontal radiographic beam angle. In this analytical study, the influence of variation of the horse's stance in the craniocaudal and lateromodial plane on hoof balance measurements as well as the influence of variation of the horizontal radiographic beam angle on dorsopalmar hoof balance measurements was examined. Distal left thoracic limb lateromedial radiographs were acquired using a standardized protocol while varying the craniocaudal stance of five horses, each selected to be sound and conformationally normal. Dorsopalmar foot radiographs were acquired while varying the lateromedial stance; and variable angle horizontal beam dorsopalmar foot radiographs were acquired while keeping the limb position constant. Analyses of measurements demonstrated that hoof pastern angle had a linear relationship (R2 = 0.89, P < 0.001) with craniocaudal stance of the horse. The relationship of joint angle and stance was greater for the distal interphalangeal joint angle (R2 = 0.89, P < 0.001) than the proximal interphalangeal joint angle (R2 = 0.65, P = 0.001). The distal phalanx angle did not change with craniocaudal stance variation. The proximal interphalangeal joint width, distal interphalangeal joint width, or distal phalanx height did not change with lateromedial stance variation, nor within a 15 degree dorsolateral to caudomedial and dorsomedial to caudolateral variation from the dorsopalmar axis. Findings indicated that positioning of the thoracic limb needs to be considered during radiographic interpretation and decision-making for corrective farriery.

Retreatment with HBV siRNA Results in Additional Reduction in HBV Antigenemia and Immune Stimulation in the AAV-HBV Mouse Model.

BACKGROUND AND AIMS: Treatment with siRNAs that target HBV has demonstrated robust declines in HBV antigens. This effect is also observed in the AAV-HBV mouse model, which was used to investigate if two cycles of GalNAc-HBV-siRNA treatment could induce deeper declines in HBsAg levels or prevent rebound, and to provide insights into the liver immune microenvironment. METHODS: C57Bl/6 mice were transduced with one of two different titers of AAV-HBV for 28 days, resulting in stable levels of HBsAg of about 103 or 105 IU/mL. Mice were treated for 12 weeks (four doses q3wk) per cycle with 3 mg/kg of siRNA-targeting HBV or an irrelevant sequence either once (single treatment) or twice (retreatment) with an 8-week treatment pause in between. Blood was collected to evaluate viral parameters. Nine weeks after the last treatment, liver samples were collected to perform phenotyping, bulk RNA-sequencing, and immunohistochemistry. RESULTS: Independent of HBsAg baseline levels, treatment with HBV-siRNA induced a rapid decline in HBsAg levels, which then plateaued before gradually rebounding 12 weeks after treatment stopped. A second cycle of HBV-siRNA treatment induced a further decline in HBsAg levels in serum and the liver, reaching undetectable levels and preventing rebound when baseline levels were 103 IU/mL. This was accompanied with a significant increase in inflammatory macrophages in the liver and significant upregulation of regulatory T-cells and T-cells expressing immune checkpoint receptors. CONCLUSIONS: Retreatment induced an additional decline in HBsAg levels, reaching undetectable levels when baseline HBsAg levels were 3log10 or less. This correlated with T-cell activation and upregulation of Trem2.

A stable hepatitis D virus-producing cell line for host target and drug discovery.

Chronic hepatitis D is the most aggressive form of chronic viral hepatitis. It is caused by super-infection of hepatitis B virus (HBV)-infected hepatocytes with hepatitis D virus (HDV). While the recent conditional approval of bulevirtide for HDV treatment offers a new therapeutic modality in Europe, there is an unmet medical need to further improve therapy. A more detailed characterization of virus-host interactions is needed for the identification of novel therapeutic targets. Addressing this need, we engineered a new stably-transformed cell line, named HuH7-2C8D, producing high titer recombinant HDV and allowing the study of viral particles morphogenesis and infectivity. Using this culture system, where viral propagation by re-infection is limited, we observed an increased accumulation of edited version of the viral genomes within secreted HDV viral particles over time that is accompanied with a decrease in viral particle infectivity. We confirmed the interaction of HDV proteins with a previously described host factor in HuH7-2C8D cells and additionally showed that these cells are suitable for co-culture assays with other cell types such as macrophages. Finally, the use of HuH7-2C8D cells allowed to confirm the dual antiviral activity of farnesyl transferase inhibitors, including the clinical candidate lonafarnib, against HDV. In conclusion, we have established an easy-to-handle cell culture model to investigate HDV replication, morphogenesis, and host interactions. HuH7-2C8D cells are also suitable for high-throughput antiviral screening assays for the development of new therapeutic strategies.

Clinicopathologic observations on laryngoplasty failure in a horse.

OBJECTIVE: To report morphologic findings associated with laryngoplasty failure in a horse. STUDY DESIGN: Clinical report. ANIMALS: A 9-year-old Thoroughbred cross gelding. METHODS: Necropsy and histopathology were performed on a horse that died peracutely during anesthetic recovery after correction of a right dorsal displacement of the ascending colon. Three weeks earlier the horse had left laryngoplasty and ventriculocordectomy. RESULTS: Dissection of the larynx revealed that the laryngoplasty suture had pulled through the muscular process of the left arytenoid cartilage, which appeared grossly normal. Histopathology of the arytenoid muscular process revealed cartilage necrosis, granulation tissue, and inflammation around the cartilage and within the cartilage failure line, and small numbers of coccoid bacteria in a minority of cartilage canals. Multifocal cardiomyopathy and pulmonary congestion, edema, and hemorrhage were also observed histologically. CONCLUSION: Death was attributed to peracute pulmonary edema associated with cardiac abnormalities and airway obstruction from laryngoplasty failure. Morphologic changes in the muscular process indicate gradual progression toward laryngoplasty failure, possibly associated with suture-induced pressure necrosis and/or microscopic low-grade postoperative infection.

1a/1b subtype profiling of nonnucleoside polymerase inhibitors of hepatitis C virus.

The RNA-dependent RNA polymerase (NS5B) of hepatitis C virus (HCV) is an unusually attractive target for drug discovery since it contains five distinct drugable sites. The success of novel antiviral therapies will require nonnucleoside inhibitors to be active in at least patients infected with HCV of subtypes 1a and 1b. Therefore, the genotypic assessment of these agents against clinical isolates derived from genotype 1-infected patients is an important prerequisite for the selection of suitable candidates for clinical development. Here we report the 1a/1b subtype profiling of polymerase inhibitors that bind at each of the four known nonnucleoside binding sites. We show that inhibition of all of the clinical isolates tested is maintained, except for inhibitors that bind at the palm-1 binding site. Subtype coverage varies across chemotypes within this class of inhibitors, and inhibition of genotype 1a improves when hydrophobic contact with the polymerase is increased. We investigated if the polymorphism of the palm-1 binding site is the sole cause of the reduced susceptibility of subtype 1a to inhibition by 1,5-benzodiazepines by using reverse genetics, X-ray crystallography, and surface plasmon resonance studies. We showed Y415F to be a key determinant in conferring resistance on subtype 1a, with this effect being mediated through an inhibitor- and enzyme-bound water molecule. Binding studies revealed that the mechanism of subtype 1a resistance is faster dissociation of the inhibitor from the enzyme.

The Hepatitis B Virus Interactome: A Comprehensive Overview.

Despite the availability of a prophylactic vaccine, chronic hepatitis B (CHB) caused by the hepatitis B virus (HBV) is a major health problem affecting an estimated 292 million people globally. Current therapeutic goals are to achieve functional cure characterized by HBsAg seroclearance and the absence of HBV-DNA after treatment cessation. However, at present, functional cure is thought to be complicated due to the presence of covalently closed circular DNA (cccDNA) and integrated HBV-DNA. Even if the episomal cccDNA is silenced or eliminated, it remains unclear how important the high level of HBsAg that is expressed from integrated HBV DNA is for the pathology. To identify therapies that could bring about high rates of functional cure, in-depth knowledge of the virus' biology is imperative to pinpoint mechanisms for novel therapeutic targets. The viral proteins and the episomal cccDNA are considered integral for the control and maintenance of the HBV life cycle and through direct interaction with the host proteome they help create the most optimal environment for the virus whilst avoiding immune detection. New HBV-host protein interactions are continuously being identified. Unfortunately, a compendium of the most recent information is lacking and an interactome is unavailable. This article provides a comprehensive review of the virus-host relationship from viral entry to release, as well as an interactome of cccDNA, HBc, and HBx.

Contrast Enhanced Computed Tomography Findings in 105 Horse Distal Extremities.

The poor soft tissue conspicuity of CT can be improved by using intra-arterial CT Angiography (CTA), and intra-articular and intra-bursal contrast enhanced CT (CTAR). This retrospective study describes a combination protocol of CT and CTA of the horse's foot, and CTAR of the distal interphalangeal joint and navicular bursa. It is hypothesized this would provide a comprehensive overview of the range and severity of distal limb pathology. Radiology reports of all horses admitted for distal limb CT over a 5 year period were reviewed. All horses with a complete four stage CT examination and radiology report with lameness isolated to the foot were included. Twenty seven imaging findings using a four grade semiquantitative severity scoring system contributing towards six main diagnostic categories were described. One hundred and five examinations on 56 horses revealed a diagnosis of navicular bone disease in 64%, deep digital flexor tendinopathy in 43%, distal interphalangeal osteoarthritis in 35%, navicular bursitis in 31%, distal interphalangeal collateral ligament desmopathy in 26%, and hoof capsule and distal phalanx pathology in 10%. Only 25% of the navicular bone disease cases were considered clinically significant. The majority of deep digital flexor tendon lesions (77%) and distal interphalangeal joint osteoarthritis (51%) were considered significant. Approximately one third of navicular bursa (37%) and collateral ligament (33%) abnormalities were considered significant. Navicular bursa abnormalities were associated with navicular bone and deep digital flexor tendon lesions. The findings support the hypothesis and the use of this protocol for evaluation of foot lameness.

Development of a cellular high-content, immunofluorescent HBV core assay to identify novel capsid assembly modulators that induce the formation of aberrant HBV core structures.

Hepatitis B Virus (HBV) core protein has multiple functions in the viral life cycle and is an attractive target for new anti-viral therapies. Capsid assembly modulators (CAMs) target the core protein and induce the formation of either morphologically normal (CAM-N) or aberrant structures (CAM-A), both devoid of genomic material. To date a diverse family of CAM-N chemotypes has been identified, but in contrast, described CAM-As are based on the heteroaryldihydropyrimidine (HAP) scaffold. We used the HBV-inducible HepG2.117 cell line with immunofluorescent labeling of HBV core to develop and validate a cellular high-content image-based assay where aggregated core structures are identified using image analysis spot texture features. Treatment with HAPs led to a dose- and time-dependent formation of aggregated core appearing as dot-like structures in the cytoplasm and nucleus. By combining a biochemical and cellular screening approach, a compound was identified as a novel non-HAP scaffold able to induce dose-dependent formation of aberrant core structures, which was confirmed by electron microscopy and native gel electrophoresis. This compound displayed anti-HBV activity in HepG2.117 cells, providing proof-of-concept for our screening approach. We believe our combined biochemical and cellular high-content screening method will aid in expanding the range of CAM-A chemotypes.

Mechanism and specificity of a symmetrical benzimidazolephenylcarboxamide helicase inhibitor.

This study examines the effects of 1-N,4-N-bis[4-(1H-benzimidazol-2-yl)phenyl]benzene-1,4-dicarboxamide ((BIP)(2)B) on the NS3 helicase encoded by the hepatitis C virus (HCV). Molecular beacon-based helicase assays were used to show that (BIP)(2)B inhibits the ability of HCV helicase to separate a variety of RNA and DNA duplexes with half-maximal inhibitory concentrations ranging from 0.7 to 5 microM, depending on the nature of the substrate. In single turnover assays, (BIP)(2)B only inhibited unwinding reactions when it was preincubated with the helicase-nucleic acid complex. (BIP)(2)B quenched NS3 intrinsic protein fluorescence with an apparent dissociation constant of 5 microM, and in the presence of (BIP)(2)B, HCV helicase did not appear to interact with a fluorescent DNA oligonucleotide. In assays monitoring HCV helicase-catalyzed ATP hydrolysis, (BIP)(2)B only inhibited helicase-catalyzed ATP hydrolysis in the presence of intermediate concentrations of RNA, suggesting RNA and (BIP)(2)B compete for the same binding site. HCV helicases isolated from various HCV genotypes were similarly sensitive to (BIP)(2)B, with half-maximal inhibitory concentrations ranging from 0.7 to 2.4 microM. (BIP)(2)B also inhibited ATP hydrolysis catalyzed by related helicases from Dengue virus, Japanese encephalitis virus, and humans. (BIP)(2)B appeared to bind the HCV and human proteins with similar affinity (K(i) = 7 and 8 microM, respectively), but it bound the flavivirus proteins up to 270 times more tightly. Results are discussed in light of a molecular model of a (BIP)(2)B-HCV helicase complex, which is unable to bind nucleic acid, thus preventing the enzyme from separating double-stranded nucleic acid.

1,5-Benzodiazepine inhibitors of HCV NS5B polymerase.

Optimization through parallel synthesis of a novel series of hepatitis C virus (HCV) NS5B polymerase inhibitors led to the identification of (R)-11-(4-benzyloxy-2-fluorophenyl)-6-hydroxy-3,3-dimethyl-10-(6-methylpyridine-2-carbonyl)-2,3,4,5,10,11-hexahydro-dibenzo[b,e][1,4]diazepin-1-one 11zc and (R)-11-(4-benzyloxy-2-fluorophenyl)-6-hydroxy-3,3-dimethyl-10-(2,5-dimethyloxazol-4-carbonyl)-2,3,4,5,10,11-hexahydro-dibenzo[b,e][1,4]diazepin-1-one 11zk as potent (replicon EC(50)=400nM and 270nM, respectively) and selective (CC(50)>20muM) inhibitors of HCV replication. These data warrant further lead-optimization efforts.

1,5-benzodiazepines, a novel class of hepatitis C virus polymerase nonnucleoside inhibitors.

The exogenous control of hepatitis C virus (HCV) replication can be mediated through the inhibition of the RNA-dependent RNA polymerase (RdRp) activity of NS5B. Small-molecule inhibitors of NS5B include nucleoside and nonnucleoside analogs. Here, we report the discovery of a novel class of HCV polymerase nonnucleoside inhibitors, 1,5-benzodiazepines (1,5-BZDs), identified by high-throughput screening of a library of small molecules. A fluorescence-quenching assay and X-ray crystallography revealed that 1,5-BZD 4a bound stereospecifically to NS5B next to the catalytic site. When introduced into replicons, mutations known to confer resistance against chemotypes that bind at this site were detrimental to inhibition by 1,5-BZD 7a. Using a panel of enzyme isolates that covered genotypes 1 to 6, we showed that compound 4a inhibited genotype 1 only. In mechanistic studies, 4a was found to inhibit the RdRp activity of NS5B noncompetitively with GTP and to inhibit the formation of the first phosphodiester bond during the polymerization cycle. The specificity for the HCV target was evaluated by profiling the 1,5-BZDs against other viral and human polymerases, as well as BZD receptors.