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.

Anti-HBV activity of the HBV capsid assembly modulator JNJ-56136379 across full-length genotype A-H clinical isolates and core site-directed mutants in vitro.

OBJECTIVES: To characterize antiviral activity of the capsid assembly modulator (CAM-N) JNJ-56136379 against HBV genotypes and variants carrying amino acid substitutions in the core protein. METHODS: Anti-HBV activity of JNJ-56136379 was investigated against a diverse panel of 53 HBV clinical isolates (genotypes A-H). The impact of core amino acid substitutions using site-directed mutants (SDMs) was assessed in a transient replication assay. RESULTS: JNJ-56136379 median 50% effective concentration (EC50) values across all genotypes were 10-33 nM versus 17 nM (genotype D reference). JNJ-56136379 remained active against isolates carrying nucleos(t)ide analogue resistance mutations (median EC50 2-25 nM) or basal core promoter (BCP) ± precore (PC) mutations (median EC50 13-20 nM) or PC mutations (median EC50 11 nM), representing activity against isolates from HBeAg-positive and -negative hepatitis B patients. Core amino acid substitutions in the CAM-binding pocket, when tested as SDMs at positions 23, 25, 30, 33, 37, 106, 110, 118, 124, 127 and 128, reduced JNJ-56136379 anti-HBV activity; EC50 fold increases ranged from 3.0 (S106T) to 85 (T33N). All substitutions were rare in a public database of >7600 HBV core sequences (frequencies 0.01%-0.3%). Nucleos(t)ide analogues retained full activity against these core SDMs. CONCLUSIONS: JNJ-56136379, a potent HBV CAM-N, currently in Phase 2 clinical development, was generally fully active against an extensive panel of genotype A-H clinical isolates, regardless of the presence of nucleos(t)ide analogue resistance or BCP/PC mutations. JNJ-56136379 activity was reduced by some core amino acid substitutions in the CAM-binding pocket.

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.

Robust isolation protocol for mouse leukocytes from blood and liver resident cells for immunology research.

Research on liver-related conditions requires a robust and efficient method to purify viable hepatocytes, lymphocytes and all other liver resident cells, such as Kupffer or liver sinusoidal endothelial cells. Here we describe a novel purification method using liver enzymatic digestion, followed by a downstream optimized purification. Using this enzymatic digestion protocol, the resident liver cells as well as viable hepatocytes could be captured, compared to the classical mechanical liver disruption method. Moreover, single-cell RNA-sequencing demonstrated higher quality lymphocyte data in downstream analyses after the liver enzymatic digestion, allowing for studying of immunological responses or changes. In order to also understand the peripheral immune landscape, a protocol for lymphocyte purification from mouse systemic whole blood was optimized, allowing for efficient removal of red blood cells. The combination of microbeads and mRNA blockers allowed for a clean blood sample, enabling robust single-cell RNA-sequencing data. These two protocols for blood and liver provide important new methodologies for liver-related studies such as NASH, hepatitis virus infections or cancer research but also for immunology where high-quality cells are indispensable for further downstream assays.

TMC647055, a potent nonnucleoside hepatitis C virus NS5B polymerase inhibitor with cross-genotypic coverage.

Hepatitis C virus (HCV) infection is a major global health burden and is associated with an increased risk of liver cirrhosis and hepatocellular carcinoma. There remains an unmet medical need for efficacious and safe direct antivirals with complementary modes of action for combination in treatment regimens to deliver a high cure rate with a short duration of treatment for HCV patients. Here we report the in vitro inhibitory activity, mode of action, binding kinetics, and resistance profile of TMC647055, a novel and potent nonnucleoside inhibitor of the HCV NS5B RNA-dependent RNA polymerase. In vitro combination studies with an HCV NS3/4A protease inhibitor demonstrated potent suppression of HCV RNA replication, confirming the potential for combination of these two classes in the treatment of chronic HCV infection. TMC647055 is a potent nonnucleoside NS5B polymerase inhibitor of HCV replication with a promising in vitro biochemical, kinetic, and virological profile that is currently undergoing clinical evaluation.

Finger loop inhibitors of the HCV NS5b polymerase. Part II. Optimization of tetracyclic indole-based macrocycle leading to the discovery of TMC647055.

Optimization of a novel series of macrocyclic indole-based inhibitors of the HCV NS5b polymerase targeting the finger loop domain led to the discovery of lead compounds exhibiting improved potency in cellular assays and superior pharmacokinetic profile. Further lead optimization performed on the most promising unsaturated-bridged subseries provided the clinical candidate 27-cyclohexyl-12,13,16,17-tetrahydro-22-methoxy-11,17-dimethyl-10,10-dioxide-2,19-methano-3,7:4,1-dimetheno-1H,11H-14,10,2,9,11,17-benzoxathiatetraazacyclo docosine-8,18(9H,15H)-dione, TMC647055 (compound 18a). This non-zwitterionic 17-membered ring macrocycle combines nanomolar cellular potency (EC(50) of 82 nM) with minimal associated cell toxicity (CC(50)>20 µM) and promising pharmacokinetic profiles in rats and dogs. TMC647055 is currently being evaluated in the clinic.

Finger-loop inhibitors of the HCV NS5b polymerase. Part 1: Discovery and optimization of novel 1,6- and 2,6-macrocyclic indole series.

Novel conformationaly constrained 1,6- and 2,6-macrocyclic HCV NS5b polymerase inhibitors, in which either the nitrogen or the phenyl ring in the C2 position of the central indole core is tethered to an acylsulfamide acid bioisostere, have been designed and tested for their anti-HCV potency. This transformational route toward non-zwitterionic finger loop-directed inhibitors led to the discovery of derivatives with improved cell potency and pharmacokinetic profile.

Antiviral activity and mode of action of TMC647078, a novel nucleoside inhibitor of the hepatitis C virus NS5B polymerase.

Chronic infection with hepatitis C virus (HCV) is a major global health burden and is associated with an increased risk of liver cirrhosis and hepatocellular carcinoma. Current therapy for HCV infection has limited efficacy, particularly against genotype 1 virus, and is hampered by a range of adverse effects. Therefore, there is a clear unmet medical need for efficacious and safe direct antiviral drugs for use in combination with current treatments to increase cure rates and shorten treatment times. The broad genotypic coverage achievable with nucleosides or nucleotides and the high genetic barrier to resistance of these compounds observed in vitro and in vivo suggest that this class of inhibitors could be a valuable component of future therapeutic regimens. Here, we report the in vitro inhibitory activity and mode of action of 2'-deoxy-2'-spirocyclopropylcytidine (TMC647078), a novel and potent nucleoside inhibitor of the HCV NS5B RNA-dependent RNA polymerase that causes chain termination of the nascent HCV RNA chain. In vitro combination studies with a protease inhibitor resulted in additive efficacy in the suppression of HCV RNA replication, highlighting the potential for the combination of these two classes in the treatment of chronic HCV infection. No cytotoxic effects were observed in various cell lines. Biochemical studies indicated that TMC647078 is phosphorylated mainly by deoxycytidine kinase (dCK) without inhibiting the phosphorylation of the natural substrate, and high levels of triphosphate were observed in Huh7 cells and in primary hepatocytes in vitro. TMC647078 is a potent novel nucleoside inhibitor of HCV replication with a promising in vitro virology and biology profile.

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.

In vitro resistance profile of the hepatitis C virus NS3/4A protease inhibitor TMC435.

TMC435 is a small-molecule inhibitor of the NS3/4A serine protease of hepatitis C virus (HCV) currently in phase 2 development. The in vitro resistance profile of TMC435 was characterized by selection experiments with HCV genotype 1 replicon cells and the genotype 2a JFH-1 system. In 80% (86/109) of the sequences from genotype 1 replicon cells analyzed, a mutation at NS3 residue D168 was observed, with changes to V or A being the most frequent. Mutations at NS3 positions 43, 80, 155, and 156, alone or in combination, were also identified. A transient replicon assay confirmed the relevance of these positions for TMC435 inhibitory activity. The change in the 50% effective concentrations (EC(50)s) observed for replicons with mutations at position 168 ranged from <10-fold for those with the D168G or D168N mutation to approximately 2,000-fold for those with the D168V or D168I mutation, compared to the EC(50) for the wild type. Of the positions identified, mutations at residue Q80 had the least impact on the activity of TMC435 (<10-fold change in EC(50)s), while greater effects were observed for some replicons with mutations at positions 43, 155, and 156. TMC435 remained active against replicons with the specific mutations observed after in vitro or in vivo exposure to telaprevir or boceprevir, including most replicons with changes at positions 36, 54, and 170 (<3-fold change in EC(50)s). Replicons carrying mutations affecting the activity of TMC435 remained fully susceptible to alpha interferon and NS5A and NS5B inhibitors. Finally, combinations of TMC435 with alpha interferon and NS5B polymerase inhibitors prevented the formation of drug-resistant replicon colonies.

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.

Comparison of drug transporter gene expression and functionality in Caco-2 cells from 10 different laboratories.

Caco-2 cells, widely used to study carrier mediated uptake and efflux mechanisms, are known to have different properties when cultured under different conditions. In this study, Caco-2 cells from 10 different laboratories were compared in terms of mRNA expression levels of 72 drug and nutrient transporters, and 17 other target genes, including drug metabolising enzymes, using real-time PCR. The rank order of the top five expressed genes was: HPT1>GLUT3>GLUT5>GST1A>OATP-B. Rank correlation showed that for most of the samples, the gene ranking was not significantly different. Functionality of transporters and the permeability of passive transport markers metoprolol (transcellular) and atenolol (paracellular) were also compared. MDR1 and PepT1 function was investigated using talinolol and Gly-Sar transport, respectively. Sulfobromophthalein (BSP) was used as a marker for MRP2 and OATP-B functionality. Atenolol permeability was more variable across laboratories than metoprolol permeability. Talinolol efflux was observed by all the laboratories, whereas only five laboratories observed significant apical uptake of Gly-Sar. Three laboratories observed significant efflux of BSP. MDR1 expression significantly correlated to the efflux ratio and net active efflux of talinolol. PepT1 mRNA levels showed significant correlation to the uptake ratio and net active uptake of Gly-Sar. MRP2 and OATP-B showed no correlation to BSP transport parameters. Heterogeneity in transporter activity may thus be due to differences in transporter expression as shown for PepT1 and MDR1 which in turn is determined by the culture conditions. Absolute expression of genes was variable indicating that small differences in culture conditions have a significant impact on gene expression, although the overall expression patterns were similar.

Synthesis and Evaluation of N-Phenyl-3-sulfamoyl-benzamide Derivatives as Capsid Assembly Modulators Inhibiting Hepatitis B Virus (HBV).

Small molecule induced hepatitis B virus (HBV) capsid assembly modulation is considered an attractive approach for new antiviral therapies against HBV. Here we describe efforts toward the discovery of a HBV capsid assembly modulator in a hit-to-lead optimization, resulting in JNJ-632, a tool compound used to further profile the mode of action. Administration of JNJ-632 (54) in HBV genotype D infected chimeric mice resulted in a 2.77 log reduction of the HBV DNA viral load.

Antiviral profiling of the capsid assembly modulator BAY41-4109 on full-length HBV genotype A-H clinical isolates and core site-directed mutants in vitro.

The HBV core protein represents an attractive target for new antiviral therapies due to its multiple functions within the viral life-cycle. Here, we report the antiviral activity of the capsid assembly modulator (CAM) BAY41-4109 and two nucleos(t)ide analogues (NAs) on a diverse panel of 54 HBV clinical isolates from genotype (GT) A-H and assessed the impact of core amino acid (aa) substitutions using site-directed mutants (SDMs). The median EC50 values of BAY41-4109 across genotypes ranged from 26 nM in GT G to 215 nM in GT F irrespective of the presence of NA resistance mutations compared to 43 nM for the GT D reference construct. Combined analyses of clinical isolates and SDMs identified aa changes at positions 29, 33 and 118 led to reduced antiviral activity of BAY41-4109 with fold changes in EC50 values of 6, 46, and 9 for D29G, T33N, and Y118F, respectively. These aa substitutions are located within the CAM binding pocket, and are expected to have an effect on CAM binding based on structural modeling. Importantly aa variations at these positions were rarely (<0.3%) observed as naturally occurring in public sequence databases. NA's remained fully active against these variants. Our study demonstrated that BAY41-4109 generally remained fully active across GT A-H clinical isolates. In addition, core aa substitutions within the CAM-binding pocket replicated in vitro and variants at positions 29, 33, and 118 were identified to reduce antiviral activity.

Plasma drug levels, genotypic resistance, and virological response to a nelfinavir plus saquinavir-containing regimen.

OBJECTIVE: To determine the importance of resistance and drug levels in the response to a dual-protease inhibitor (PI) combination. METHODS: Prospective study of 62 HIV-positive patients who switched to a salvage regimen including nelfinavir plus saquinavir. Virological response was defined as a decrease in viraemia > 0.5 log10 after 24 weeks. Optimal PI levels were defined as those above the protein binding-corrected 95% inhibitory concentration (IC95), as estimated in the presence of 50% human serum. RESULTS: Baseline median HIV load was 4.78 log10 copies/ml. The median number of mutations in the protease gene was nine (range, 2-25), predominantly at residues 82 (52%), and 90 (40%). After 24 weeks, 45% of patients had responded and 19% were < 50 copies/ml. A higher number of mutations in the protease gene (12 versus 8;P = 0.001), and the L90M mutation (36% versus 67%; P = 0.001) were associated with treatment failure. Trough levels of nelfinavir and saquinavir were two- and fivefold, respectively, greater than those reached when used as the only PI (2480 and 260 ng/ml, respectively), and they were above the estimated protein-corrected IC95 in 96% and 32% of cases. Thus, the Cmin : IC95 ratio ranged from 0.1 to 10 for nelfinavir and from 0.12 to 3.24 for saquinavir. Suboptimal PI levels were associated with a poorer response, but there was no correlation between optimal drug levels and a better response. CONCLUSION: Genotypic resistance predicts the virological response to a nelfinavir-saquinavir salvage regimen. Our data suggest that higher than optimal drug levels could be necessary to control the replication of many PI-resistant viruses.

Discovery and early development of TMC647055, a non-nucleoside inhibitor of the hepatitis C virus NS5B polymerase.

Structure-based macrocyclization of a 6-carboxylic acid indole chemotype has yielded potent and selective finger-loop inhibitors of the hepatitis C virus (HCV) NS5B polymerase. Lead optimization in conjunction with in vivo evaluation in rats identified several compounds showing (i) nanomolar potency in HCV replicon cells, (ii) limited toxicity and off-target activities, and (iii) encouraging preclinical pharmacokinetic profiles characterized by high liver distribution. This effort culminated in the identification of TMC647055 (10a), a nonzwitterionic 17-membered-ring macrocycle characterized by high affinity, long polymerase residence time, and broad genotypic coverage. In vitro results of the combination of 10a with the HCV protease inhibitor TMC435 (simeprevir) supported an evaluation of this combination in patients with regard to virus suppression and resistance emergence. In a phase 1b trial with HCV genotype 1-infected patients, 10a was considered to be safe and well-tolerated and demonstrated potent antiviral activity, which was further enhanced in a combination study with TMC435.

Identification of HIV-1 reverse transcriptase inhibitors using a scintillation proximity assay.

The human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) converts the viral single-stranded RNA into double-stranded DNA. The inhibition of reverse transcription in the viral life cycle has proven its efficacy as a clinically relevant antiviral target, but the appearance of resistance mutations remains a major cause of treatment failure and stresses the continuous need for new antiviral compounds. In this chapter, we describe an HIV-1 RT scintillation proximity assay (SPA) to identify inhibitors of the RT. The assay uses an RNA/DNA (poly(rA)/oligo(dT)) template/primer bound to SPA beads, which contain scintillant. Reverse transcriptase extends the primer by incorporating [(3)H]dTTP and dTTP, which results in light production by the scintillant in the bead. Compounds that inhibit reverse transcriptase will prevent the incorporation of tritiated dTTP resulting in a reduction of emitted light compared to the untreated controls.

Indolopyridones inhibit human immunodeficiency virus reverse transcriptase with a novel mechanism of action.

We have discovered a novel class of human immunodeficiency virus (HIV) reverse transcriptase (RT) inhibitors that block the polymerization reaction in a mode distinct from those of the nucleoside or nucleotide RT inhibitors (NRTIs) and nonnucleoside RT inhibitors (NNRTIs). For this class of indolopyridone compounds, steady-state kinetics revealed competitive inhibition with respect to the nucleotide substrate. Despite substantial structural differences with classical chain terminators or natural nucleotides, these data suggest that the nucleotide binding site of HIV RT may accommodate this novel class of RT inhibitors. To test this hypothesis, we have studied the mechanism of action of the prototype compound indolopyridone-1 (INDOPY-1) using a variety of complementary biochemical tools. Time course experiments with heteropolymeric templates showed "hot spots" for inhibition following the incorporation of pyrimidines (T>C). Moreover, binding studies and site-specific footprinting experiments revealed that INDOPY-1 traps the complex in the posttranslocational state, preventing binding and incorporation of the next complementary nucleotide. The novel mode of action translates into a unique resistance profile. While INDOPY-1 susceptibility is unaffected by mutations associated with NNRTI or multidrug NRTI resistance, mutations M184V and Y115F are associated with decreased susceptibility, and mutation K65R confers hypersusceptibility to INDOPY-1. This resistance profile provides additional evidence for active site binding. In conclusion, this class of indolopyridones can occupy the nucleotide binding site of HIV RT by forming a stable ternary complex whose stability is mainly dependent on the nature of the primer 3' end.