Discovery of a first-in-class orally available HBV cccDNA inhibitor.

BACKGROUND & AIMS: The persistence of covalently closed circular DNA (cccDNA) in infected hepatocytes is the major barrier preventing viral eradication with existing therapies in patients with chronic hepatitis B. Therapeutic agents that can eliminate cccDNA are urgently needed to achieve viral eradication and thus HBV cure. METHODS: A phenotypic assay with HBV-infected primary human hepatocytes (PHHs) was employed to screen for novel cccDNA inhibitors. A HBVcircle mouse model and a uPA-SCID (urokinase-type plasminogen activator-severe combined immunodeficiency) humanized liver mouse model were used to evaluate the anti-HBV efficacy of the discovered cccDNA inhibitors. RESULTS: Potent and dose-dependent reductions in extracellular HBV DNA, HBsAg, and HBeAg levels were achieved upon the initiation of ccc_R08 treatment two days after the HBV infection of PHHs. More importantly, the level of cccDNA was specifically reduced by ccc_R08, while it did not obviously affect mitochondrial DNA. Additionally, ccc_R08 showed no significant cytotoxicity in PHHs or in multiple proliferating cell lines. The twice daily oral administration of ccc_R08 to HBVcircle model mice, which contained surrogate cccDNA molecules, significantly decreased the serum levels of HBV DNA and antigens, and these effects were sustained during the off-treatment follow-up period. Moreover, at the end of follow-up, the levels of surrogate cccDNA molecules in the livers of ccc_R08-treated HBVcircle mice were reduced to below the lower limit of quantification. CONCLUSIONS: We have discovered a small-molecule cccDNA inhibitor that reduces HBV cccDNA levels. cccDNA inhibitors potentially represent a new approach to completely cure patients chronically infected with HBV. IMPACT AND IMPLICATIONS: Covalently closed circular DNA (cccDNA) persistence in HBV-infected hepatocytes is the root cause of chronic hepatitis B. We discovered a novel small-molecule cccDNA inhibitor that can specifically reduce cccDNA levels in HBV-infected hepatocytes. This type of molecule could offer a new approach to completely cure patients chronically infected with HBV.

Discovery of Novel cccDNA Reducers toward the Cure of Hepatitis B Virus Infection.

Chronic hepatitis B virus (HBV) infection is a worldwide disease that causes thousands of deaths per year. Currently, there is no therapeutic that can completely cure already infected HBV patients due to the inability of humans to eliminate covalently closed circular DNA (cccDNA), which serves as the template to (re)initiate an infection even after prolonged viral suppression. Through phenotypic screening, we discovered xanthone series hits as novel HBV cccDNA reducers, and subsequent structure optimization led to the identification of a lead compound with improved antiviral activity and pharmacokinetic profiles. A representative compound 59 demonstrated good potency and oral bioavailability with no cellular toxicity. In an HBVcircle mouse model, compound 59 showed excellent efficacy in significantly reducing HBV antigens, DNA, and intrahepatic cccDNA levels.

In Silico and In Vitro Comparison of HIV-1 Subtypes B and CRF02_AG Integrases Susceptibility to Integrase Strand Transfer Inhibitors.

Most antiretroviral medical treatments were developed and tested principally on HIV-1 B nonrecombinant strain, which represents less than 10% of the worldwide HIV-1-infected population. HIV-1 circulating recombinant form CRF02_AG is prevalent in West Africa and is becoming more frequent in other countries. Previous studies suggested that the HIV-1 polymorphisms might be associated to variable susceptibility to antiretrovirals. This study is pointed to compare the susceptibility to integrase (IN) inhibitors of HIV-1 subtype CRF02_AG IN respectively to HIV-1 B. Structural models of B and CRF02_AG HIV-1 INs as unbound enzymes and in complex with the DNA substrate were built by homology modeling. IN inhibitors-raltegravir (RAL), elvitegravir (ELV) and L731,988-were docked onto the models, and their binding affinity for both HIV-1 B and CRF02_AG INs was compared. CRF02_AG INs were cloned and expressed from plasma of integrase strand transfer inhibitor (INSTI)-naïve infected patients. Our in silico and in vitro studies showed that the sequence variations between the INs of CRF02_AG and B strains did not lead to any notable difference in the structural features of the enzyme and did not impact the susceptibility to the IN inhibitors. The binding modes and affinities of INSTI inhibitors to B and CRF02_AG INs were found to be similar. Although previous studies suggested that several naturally occurring variations of CRF02_AG IN might alter either IN/vDNA interactions or INSTIs binding, our study demonstrate that these variations do affect neither IN activity nor its susceptibility to INSTIs.

G140S/Q148R and N155H mutations render HIV-2 Integrase resistant to raltegravir whereas Y143C does not.

BACKGROUND: HIV-2 is endemic in West Africa and has spread throughout Europe. However, the alternatives for HIV-2-infected patients are more limited than for HIV-1. Raltegravir, an integrase inhibitor, is active against wild-type HIV-2, with a susceptibility to this drug similar to that of HIV-1, and is therefore a promising option for use in the treatment of HIV-2-infected patients. Recent studies have shown that HIV-2 resistance to raltegravir involves one of three resistance mutations, N155H, Q148R/H and Y143C, previously identified as resistance determinants in the HIV-1 integrase coding sequence. The resistance of HIV-1 IN has been confirmed in vitro for mutated enzymes harboring these mutations, but no such confirmation has yet been obtained for HIV-2. RESULTS: The integrase coding sequence was amplified from plasma samples collected from ten patients infected with HIV-2 viruses, of whom three RAL-naïve and seven on RAL-based treatment at the time of virological failure. The genomes of the resistant strains were cloned and three patterns involving N155H, G140S/Q148R or Y143C mutations were identified. Study of the susceptibility of integrases, either amplified from clinical isolates or obtained by mutagenesis demonstrated that mutations at positions 155 and 148 render the integrase resistant to RAL. The G140S mutation conferred little resistance, but compensated for the catalytic defect due to the Q148R mutation. Conversely, Y143C alone did not confer resistance to RAL unless E92Q is also present. Furthermore, the introduction of the Y143C mutation into the N155H resistant background decreased the resistance level of enzymes containing the N155H mutation. CONCLUSION: This study confirms that HIV-2 resistance to RAL is due to the N155H, G140S/Q148R or E92Q/Y143C mutations. The N155H and G140S/Q148R mutations make similar contributions to resistance in both HIV-1 and HIV-2, but Y143C is not sufficient to account for the resistance of HIV-2 genomes harboring this mutation. For Y143C to confer resistance in vitro, it must be accompanied by E92Q, which therefore plays a more important role in the HIV-2 context than in the HIV-1 context. Finally, the Y143C mutation counteracts the resistance conferred by the N155H mutation, probably accounting for the lack of detection of these mutations together in a single genome.