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.

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.

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.

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.

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.

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.

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.

Novel antibiotics targeting respiratory ATP synthesis in Gram-positive pathogenic bacteria.

Emergence of drug-resistant bacteria represents a high, unmet medical need, and discovery of new antibacterials acting on new bacterial targets is strongly needed. ATP synthase has been validated as an antibacterial target in Mycobacterium tuberculosis, where its activity can be specifically blocked by the diarylquinoline TMC207. However, potency of TMC207 is restricted to mycobacteria with little or no effect on the growth of other Gram-positive or Gram-negative bacteria. Here, we identify diarylquinolines with activity against key Gram-positive pathogens, significantly extending the antibacterial spectrum of the diarylquinoline class of drugs. These compounds inhibited growth of Staphylococcus aureus in planktonic state as well as in metabolically resting bacteria grown in a biofilm culture. Furthermore, time-kill experiments showed that the selected hits are rapidly bactericidal. Drug-resistant mutations were mapped to the ATP synthase enzyme, and biochemical analysis as well as drug-target interaction studies reveal ATP synthase as a target for these compounds. Moreover, knockdown of the ATP synthase expression strongly suppressed growth of S. aureus, revealing a crucial role of this target in bacterial growth and metabolism. Our data represent a proof of principle for using the diarylquinoline class of antibacterials in key Gram-positive pathogens. Our results suggest that broadening the antibacterial spectrum for this chemical class is possible without drifting off from the target. Development of the diarylquinolines class may represent a promising strategy for combating Gram-positive pathogens.

Selectivity of TMC207 towards mycobacterial ATP synthase compared with that towards the eukaryotic homologue.

The diarylquinoline TMC207 kills Mycobacterium tuberculosis by specifically inhibiting ATP synthase. We show here that human mitochondrial ATP synthase (50% inhibitory concentration [IC(50)] of >200 microM) displayed more than 20,000-fold lower sensitivity for TMC207 compared to that of mycobacterial ATP synthase (IC(50) of 10 nM). Also, oxygen consumption in mouse liver and bovine heart mitochondria showed very low sensitivity for TMC207. These results suggest that TMC207 may not elicit ATP synthesis-related toxicity in mammalian cells. ATP synthase, although highly conserved between prokaryotes and eukaryotes, may still qualify as an attractive antibiotic target.

Diarylquinolines are bactericidal for dormant mycobacteria as a result of disturbed ATP homeostasis.

An estimated one-third of the world population is latently infected with Mycobacterium tuberculosis. These nonreplicating, dormant bacilli are tolerant to conventional anti-tuberculosis drugs, such as isoniazid. We recently identified diarylquinoline R207910 (also called TMC207) as an inhibitor of ATP synthase with a remarkable activity against replicating mycobacteria. In the present study, we show that R207910 kills dormant bacilli as effectively as aerobically grown bacilli with the same target specificity. Despite a transcriptional down-regulation of the ATP synthase operon and significantly lower cellular ATP levels, we show that dormant mycobacteria do possess residual ATP synthase enzymatic activity. This activity is blocked by nanomolar concentrations of R207910, thereby further reducing ATP levels and causing a pronounced bactericidal effect. We conclude that this residual ATP synthase activity is indispensable for the survival of dormant mycobacteria, making it a promising drug target to tackle dormant infections. The unique dual bactericidal activity of diarylquinolines on dormant as well as replicating bacterial subpopulations distinguishes them entirely from the current anti-tuberculosis drugs and underlines the potential of R207910 to shorten tuberculosis treatment.

Diarylquinolines target subunit c of mycobacterial ATP synthase.

The diarylquinoline R207910 (TMC207) is a promising candidate in clinical development for the treatment of tuberculosis. Though R207910-resistant mycobacteria bear mutations in ATP synthase, the compound's precise target is not known. Here we establish by genetic, biochemical and binding assays that the oligomeric subunit c (AtpE) of ATP synthase is the target of R207910. Thus targeting energy metabolism is a new, promising approach for antibacterial drug discovery.