Di-fluoro azepane HBV capsid assembly modulators.

The protein that forms the inner shell of the HBV virus, known as the capsid core protein, plays a crucial role in allowing chronic HBV infections to persist. Studies have shown that disrupting the assembly of the capsid can effectively combat the virus, and small molecule drugs that target the HBV capsid assembly modulator (CAM) process have been successful in clinical trials. Herein is described a distinct series of di-fluoro azepane CAMs with exceptional potency, pharmacokinetic, and solubility properties.

Diazepinone HBV capsid assembly modulators.

The HBV capsid core protein serves a number of important functions in the viral life cycle enabling chronic HBV infection to persist, and therefore is a promising drug target. Interfering with capsid assembly has shown efficacy in clinical trials with small molecule capsid assembly modulators (CAMs). Herein is described the further optimization of a progressive series of diazepinone HBV CAMs.

Oxadiazepinone HBV capsid assembly modulators.

The HBV core protein serves multiple essential functions in the viral life cycle that enable chronic HBV infection to persist, and as such, represents a promising drug target. Modulation of the HBV capsid assembly has shown efficacy in early clinical trials through use of small molecule capsid assembly modulators (CAMs). Herein is described the evolution and SAR of a novel pyrazolo piperidine lead series into advanced oxadiazepinone HBV CAMs.

Identification of a new class of HBV capsid assembly modulator.

The HBV core protein is a druggable target of interest due to the multiple essential functions in the HBV life cycle to enable chronic HBV infection. The core protein oligomerizes to form the viral capsid, and modulation of the HBV capsid assembly has shown efficacy in clinical trials. Herein is described the identification and hit to lead SAR of a novel series of pyrazolo piperidine HBV capsid assembly modulators.

P450s under Restriction (PURE) Screen Using HepaRG and Primary Human Hepatocytes for Discovery of Novel HBV Antivirals.

Herein is reported a novel screening paradigm PURE (P450s under restriction) for the identification and optimization of hits as part of a hepatitis B virus (HBV) antiviral discovery program. To closely represent in vivo hepatocytes, differentiated HepaRG cells (dHRGs) and primary human hepatocytes (PHHs) were used as the basis for an HBV infection system. However, a significant challenge arose during potency evaluation in using cultured dHRGs and PHHs as screening platforms because, as with hepatocytes in vivo, these cells express active cytochrome P450 enzymes and thus can metabolize test compounds. The observed antiviral effects may be the cumulative result of a dynamic pool of parent compound and metabolites thus confounding structure activity relationship (SAR) interpretation and subsequent optimization design initiatives. We show here that PURE methodology restricts metabolism of HBV-infected dHRGs and PHHs and thus provides highly informative potency data for decision-making on key representative antiviral compounds.

SAR studies in the sulfonyl carboxamide class of HBV capsid assembly modulators.

The HBV core protein has multiple essential functions in the HBV life cycle to enable chronic HBV infection. The core protein oligomerizes to form the viral capsid, and modulation of the HBV capsid assembly process has shown clinical efficacy in early clinical trials. Herein is described the SAR exploration of NVR 3-778, the first clinical compound in the sulfonyl carboxamide class.

Preclinical Characterization of NVR 3-778, a First-in-Class Capsid Assembly Modulator against Hepatitis B Virus.

NVR 3-778 is the first capsid assembly modulator (CAM) that has demonstrated antiviral activity in hepatitis B virus (HBV)-infected patients. NVR 3-778 inhibited the generation of infectious HBV DNA-containing virus particles with a mean antiviral 50% effective concentration (EC50) of 0.40 µM in HepG2.2.15 cells. The antiviral profile of NVR 3-778 indicates pan-genotypic antiviral activity and a lack of cross-resistance with nucleos(t)ide inhibitors of HBV replication. The combination of NVR 3-778 with nucleos(t)ide analogs in vitro resulted in additive or synergistic antiviral activity. Mutations within the hydrophobic pocket at the dimer-dimer interface of the core protein could confer resistance to NVR 3-778, which is consistent with the ability of the compound to bind to core and to induce capsid assembly. By targeting core, NVR 3-778 inhibits pregenomic RNA encapsidation, viral replication, and the production of HBV DNA- and HBV RNA-containing particles. NVR 3-778 also inhibited de novo infection and viral replication in primary human hepatocytes with EC50 values of 0.81 µM against HBV DNA and between 3.7 and 4.8 µM against the production of HBV antigens and intracellular HBV RNA. NVR 3-778 showed favorable pharmacokinetics and safety in animal species, allowing serum levels in excess of 100 µM to be achieved in mice and, thus, enabling efficacy studies in vivo The overall preclinical profile of NVR 3-778 predicts antiviral activity in vivo and supports its further evaluation for safety, pharmacokinetics, and antiviral activity in HBV-infected patients.

Hepatitis B Virus Capsid Assembly Modulators, but Not Nucleoside Analogs, Inhibit the Production of Extracellular Pregenomic RNA and Spliced RNA Variants.

The hepatitis B virus (HBV) core protein serves multiple essential functions in the viral life cycle, and antiviral agents that target the core protein are being developed. Capsid assembly modulators (CAMs) are compounds that target core and misdirect capsid assembly, resulting in the suppression of HBV replication and virion production. Besides HBV DNA, circulating HBV RNA has been detected in patient serum and can be associated with the treatment response. Here we studied the effect of HBV CAMs on the production of extracellular HBV RNA using infected HepaRG cells and primary human hepatocytes. Representative compounds from the sulfonamide carboxamide and heteroaryldihydropyrimidine series of CAMs were evaluated and compared to nucleos(t)ide analogs as inhibitors of the viral polymerase. The results showed that CAMs blocked extracellular HBV RNA with efficiencies similar to those with which they blocked pregenomic RNA (pgRNA) encapsidation, HBV DNA replication, and Dane particle production. Nucleos(t)ide analogs inhibited viral replication and virion production but not encapsidation or production of extracellular HBV RNA. Profiling of HBV RNA from both culture supernatants and patient serum showed that extracellular viral RNA consisted of pgRNA and spliced pgRNA variants with an internal deletion(s) but still retained the sequences at both the 5' and 3' ends. Similar variants were detected in the supernatants of infected cells with and without nucleos(t)ide analog treatment. Overall, our data demonstrate that HBV CAMs represent direct antiviral agents with a profile differentiated from that of nucleos(t)ide analogs, including the inhibition of extracellular pgRNA and spliced pgRNA.

Viral genome imaging of hepatitis C virus to probe heterogeneous viral infection and responses to antiviral therapies.

Hepatitis C virus (HCV) is a positive single-stranded RNA virus of enormous global health importance, with direct-acting antiviral therapies replacing an immunostimulatory interferon-based regimen. The dynamics of HCV positive and negative-strand viral RNAs (vRNAs) under antiviral perturbations have not been studied at the single-cell level, leaving a gap in our understanding of antiviral kinetics and host-virus interactions. Here, we demonstrate quantitative imaging of HCV genomes in multiple infection models, and multiplexing of positive and negative strand vRNAs and host antiviral RNAs. We capture the varying kinetics with which antiviral drugs with different mechanisms of action clear HCV infection, finding the NS5A inhibitor daclatasvir to induce a rapid decline in negative-strand viral RNAs. We also find that the induction of host antiviral genes upon interferon treatment is positively correlated with viral load in single cells. This study adds smFISH to the toolbox available for analyzing the treatment of RNA virus infections.

High-resolution crystal structure of a hepatitis B virus replication inhibitor bound to the viral core protein.

The hepatitis B virus (HBV) core protein is essential for HBV replication and an important target for antiviral drug discovery. We report the first, to our knowledge, high-resolution crystal structure of an antiviral compound bound to the HBV core protein. The compound NVR-010-001-E2 can induce assembly of the HBV core wild-type and Y132A mutant proteins and thermostabilize the proteins with a Tm increase of more than 10 °C. NVR-010-001-E2 binds at the dimer-dimer interface of the core proteins, forms a new interaction surface promoting protein-protein interaction, induces protein assembly, and increases stability. The impact of naturally occurring core protein mutations on antiviral activity correlates with NVR-010-001-E2 binding interactions determined by crystallography. The crystal structure provides understanding of a drug efficacy mechanism related to the induction and stabilization of protein-protein interactions and enables structure-guided design to improve antiviral potency and drug-like properties.

SEC14L2 enables pan-genotype HCV replication in cell culture.

Since its discovery in 1989, efforts to grow clinical isolates of the hepatitis C virus (HCV) in cell culture have met with limited success. Only the JFH-1 isolate has the capacity to replicate efficiently in cultured hepatoma cells without cell culture-adaptive mutations. We hypothesized that cultured cells lack one or more factors required for the replication of clinical isolates. To identify the missing factors, we transduced Huh-7.5 human hepatoma cells with a pooled lentivirus-based human complementary DNA (cDNA) library, transfected the cells with HCV subgenomic replicons lacking adaptive mutations, and selected for stable replicon colonies. This led to the identification of a single cDNA, SEC14L2, that enabled RNA replication of diverse HCV genotypes in several hepatoma cell lines. This effect was dose-dependent, and required the continuous presence of SEC14L2. Full-length HCV genomes also replicated and produced low levels of infectious virus. Remarkably, SEC14L2-expressing Huh-7.5 cells also supported HCV replication following inoculation with patient sera. Mechanistic studies suggest that SEC14L2 promotes HCV infection by enhancing vitamin E-mediated protection against lipid peroxidation. This provides a foundation for development of in vitro replication systems for all HCV isolates, creating a useful platform to dissect the mechanisms by which cell culture-adaptive mutations act.

Hepatitis C virus genotype 5a subgenomic replicons for evaluation of direct-acting antiviral agents.

Hepatitis C virus (HCV) exists as six major genotypes that differ in geographical distribution, pathogenesis, and response to antiviral therapy. In vitro replication systems for all HCV genotypes except genotype 5 have been reported. In this study, we recovered genotype 5a full-length genomes from four infected voluntary blood donors in South Africa and established a G418-selectable subgenomic replicon system using one of these strains. The replicon derived from the wild-type sequence failed to replicate in Huh-7.5 cells. However, the inclusion of the S2205I amino acid substitution, a cell culture-adaptive change originally described for a genotype 1b replicon, resulted in a small number of G418-resistant cell colonies. HCV RNA replication in these cells was confirmed by quantification of viral RNA and detection of the nonstructural protein NS5A. Sequence analysis of the viral RNAs isolated from multiple independent cell clones revealed the presence of several nonsynonymous mutations, which were localized mainly in the NS3 protein. These mutations, when introduced back into the parental backbone, significantly increased colony formation. To facilitate convenient monitoring of HCV RNA replication levels, the mutant with the highest replication level was further modified to express a fusion protein of firefly luciferase and neomycin phosphotransferase. Using such replicons from genotypes 1a, 1b, 2a, 3a, 4a, and 5a, we compared the effects of various HCV inhibitors on their replication. In conclusion, we have established an in vitro replication system for HCV genotype 5a, which will be useful for the development of pan-genotype anti-HCV compounds.

Use of 2'-spirocyclic ethers in HCV nucleoside design.

Conformationally restricted 2'-spironucleosides and their prodrugs were synthesized as potential anti-HCV agents. Although the replicon activity of the new agents containing pyrimidine bases was modest, the triphosphate of a 2'-oxetane cytidine analogue demonstrated potent intrinsic biochemical activity against the NS5B polymerase, with IC50 = 8.48 µM. Activity against NS5B bearing the S282T mutation was reduced. Phosphoramidate prodrugs of a 2'-oxetane 2-amino-6-O-methyl-purine nucleoside demonstrated potent anti-HCV activity in vitro, and the corresponding triphosphate retained similar potent activity against both wild-type and S282T HCV NS5B polymerase.

Discovery of novel HCV inhibitors: synthesis and biological activity of 6-(indol-2-yl)pyridine-3-sulfonamides targeting hepatitis C virus NS4B.

A novel series of 6-(indol-2-yl)pyridine-3-sulfonamides was prepared and evaluated for their ability to inhibit HCV RNA replication in the HCV replicon cell culture assay. Preliminary optimization of this series furnished compounds with low nanomolar potency against the HCV genotype 1b replicon. Among these, compound 8c has identified as a potent HCV replicon inhibitor (EC50=4 nM) with a selectivity index with respect to cellular GAPDH of more than 2500. Further, compound 8c had a good pharmacokinetic profile in rats with an IV half-life of 6h and oral bioavailability (F) of 62%. Selection of HCV replicon resistance identified an amino acid substitution in HCV NS4B that confers resistance to these compounds. These compounds hold promise as a new chemotype with anti-HCV activity mediated through an underexploited viral target.

ß-D-2'-α-F-2'-ß-C-Methyl-6-O-substituted 3',5'-cyclic phosphate nucleotide prodrugs as inhibitors of hepatitis C virus replication: a structure-activity relationship study.

The 3',5'-cyclic phosphate prodrug 9-[ß-d-2'-deoxy-2'-α-fluoro-2'-ß-C-methylribofuranosyl]-2-amino-6-ethoxypurine, PSI-352938 1, has demonstrated promising anti-HCV efficacy in vitro and in human clinical trials. A structure-activity relationship study of the nucleoside 3',5'-cyclic phosphate series of ß-d-2'-deoxy-2'-α-fluoro-2'-ß-C-methylribofuranosyl nucleoside prodrugs was undertaken and the anti-HCV activity and in vitro safety profile were assessed. Cycloalkyl 3',5'-cyclic phosphate prodrugs were shown to be significantly more potent as inhibitors of HCV replication than branched and straight chain alkyl 3',5'-cyclic phosphate prodrugs. No cytotoxicity and mitochondrial toxicity for prodrugs 12, 13 and 19 were observed at concentrations up to 100 µm in vitro. Cycloalkyl esters of 3',5'-cyclic phosphate nucleotide prodrugs demonstrated the ability to produce high levels of active triphosphate in clone-A cells and primary human hepatocytes. Compounds 12, 13 and 19 also demonstrated the ability to effectively deliver in vivo high levels of active nucleoside phosphates to rat liver.

A 2'-deoxy-2'-fluoro-2'-C-methyl uridine cyclopentyl carbocyclic analog and its phosphoramidate prodrug as inhibitors of HCV NS5B polymerase.

The 2 '-deoxy-2 '-fluoro-2 '-C-methyluridine nucleotide prodrug, PSI-7851 and its single diastereomer PSI-7977 have displayed potent antiviral activity against hepatitis C virus in clinical trials, and PSI-7977 is currently in Phase III studies. As part of our SAR study of the 2 '-deoxy-2 '-fluoro-2 '- C-methyl class of nucleosides, we prepared the cyclopentyl carbocyclic uridine analog 11 and its phosphoramidate prodrug 15. Both 11 and 15 were shown not to inhibit HCV replication. This lack of activity might be attributed to the inability of the monophosphate to be converted to the corresponding diphosphate or triphosphate or the inactivity of triphosphate of 11 as an inhibitor of the polymerase.

Genotype and subtype profiling of PSI-7977 as a nucleotide inhibitor of hepatitis C virus.

PSI-7977, a prodrug of 2'-F-2'-C-methyluridine monophosphate, is the purified diastereoisomer of PSI-7851 and is currently being investigated in phase 3 clinical trials for the treatment of hepatitis C. In this study, we profiled the activity of PSI-7977 and its ability to select for resistance using a number of different replicon cells. Results showed that PSI-7977 was active against genotype (GT) 1a, 1b, and 2a (strain JFH-1) replicons and chimeric replicons containing GT 2a (strain J6), 2b, and 3a NS5B polymerase. Cross-resistance studies using GT 1b replicons confirmed that the S282T change conferred resistance to PSI-7977. Subsequently, we evaluated the ability of PSI-7977 to select for resistance using GT 1a, 1b, and 2a (JFH-1) replicon cells. S282T was the common mutation selected among all three genotypes, but while it conferred resistance to PSI-7977 in GT 1a and 1b, JFH-1 GT 2a S282T showed only a very modest shift in 50% effective concentration (EC(50)) for PSI-7977. Sequence analysis of the JFH-1 NS5B region indicated that additional amino acid changes were selected both prior to and after the emergence of S282T. These include T179A, M289L, I293L, M434T, and H479P. Residues 179, 289, and 293 are located within the finger and palm domains, while 434 and 479 are located on the surface of the thumb domain. Data from the JFH-1 replicon variants showed that amino acid changes within the finger and palm domains together with S282T were required to confer resistance to PSI-7977, while the mutations on the thumb domain serve to enhance the replication capacity of the S282T replicons.

Synthesis of stable isotope labeled analogs of the anti-hepatitis C virus nucleotide prodrugs PSI-7977 and PSI-352938.

In order to support bioanalytical LC/MS method development and plasma sample analysis in preclinical and clinical studies of the anti-hepatitis C-virus nucleotides, PSI-7977 and PSI-352938, the corresponding stable isotope labeled forms were prepared. These labeled compounds were prepared by addition reaction of the freshly prepared Grignard reagent (13)CD(3)MgI to the corresponding 2 '-ketone nucleosides followed by fluorination of the resulting carbinol with DAST. As expected, these 2 '-C-(trideuterated-(13)C-methyl) nucleotide prodrugs showed similar anti-HCV activity to that of the corresponding unlabeled ones.

Hepatitis C virus nucleotide inhibitors PSI-352938 and PSI-353661 exhibit a novel mechanism of resistance requiring multiple mutations within replicon RNA.

PSI-352938, a cyclic phosphate nucleotide, and PSI-353661, a phosphoramidate nucleotide, are prodrugs of ß-D-2'-deoxy-2'-α-fluoro-2'-ß-C-methylguanosine-5'-monophosphate. Both compounds are metabolized to the same active 5'-triphosphate, PSI-352666, which serves as an alternative substrate inhibitor of the NS5B RNA-dependent RNA polymerase during HCV replication. PSI-352938 and PSI-353661 retained full activity against replicons containing the S282T substitution, which confers resistance to certain 2'-substituted nucleoside/nucleotide analogs. PSI-352666 was also similarly active against both wild-type and S282T NS5B polymerases. In order to identify mutations that confer resistance to these compounds, in vitro selection studies were performed using HCV replicon cells. While no resistant genotype 1a or 1b replicons could be selected, cells containing genotype 2a JFH-1 replicons cultured in the presence of PSI-352938 or PSI-353661 developed resistance to both compounds. Sequencing of the NS5B region identified a number of amino acid changes, including S15G, R222Q, C223Y/H, L320I, and V321I. Phenotypic evaluation of these mutations indicated that single amino acid changes were not sufficient to significantly reduce the activity of PSI-352938 and PSI-353661. Instead, a combination of three amino acid changes, S15G/C223H/V321I, was required to confer a high level of resistance. No cross-resistance exists between the 2'-F-2'-C-methylguanosine prodrugs and other classes of HCV inhibitors, including 2'-modified nucleoside/-tide analogs such as PSI-6130, PSI-7977, INX-08189, and IDX-184. Finally, we determined that in genotype 1b replicons, the C223Y/H mutation failed to support replication, and although the A15G/C223H/V321I triple mutation did confer resistance to PSI-352938 and PSI-353661, this mutant replicated at only about 10% efficiency compared to the wild type.

Activity and the metabolic activation pathway of the potent and selective hepatitis C virus pronucleotide inhibitor PSI-353661.

PSI-353661, a phosphoramidate prodrug of 2'-deoxy-2'-fluoro-2'-C-methylguanosine-5'-monophosphate, is a highly active inhibitor of genotype 1a, 1b, and 2a HCV RNA replication in the replicon assay and of genotype 1a and 2a infectious virus replication. PSI-353661 is active against replicons harboring the NS5B S282T or S96T/N142T amino acid alterations that confer decreased susceptibility to nucleoside/tide analogs as well as mutations that confer resistance to non-nucleoside inhibitors of NS5B. Replicon clearance studies show that PSI-353661 was able to clear cells of HCV replicon RNA and prevent a rebound in replicon RNA. PSI-353661 showed no toxicity toward bone marrow stem cells or mitochondrial toxicity. The metabolism to the active 5'-triphosphate involves hydrolysis of the carboxyl ester by cathepsin A (Cat A) and carboxylesterase 1 (CES1) followed by a putative nucleophilic attack on the phosphorus by the carboxyl group resulting in the elimination of phenol and the alaninyl phosphate metabolite, PSI-353131. Histidine triad nucleotide-binding protein 1 (Hint 1) then removes the amino acid moiety, which is followed by hydrolysis of the methoxyl group at the O(6)-position of the guanine base by adenosine deaminase-like protein 1 (ADAL1) to give 2'-deoxy-2'-fluoro-2'-C-methylguanosine-5'-monophosphate. The monophosphate is phosphorylated to the diphosphate by guanylate kinase. Nucleoside diphosphate kinase is the primary enzyme involved in phosphorylation of the diphosphate to the active triphosphate, PSI-352666. PSI-352666 is equally active against wild-type NS5B and NS5B containing the S282T amino acid alteration.