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 a Novel Respiratory Syncytial Virus Replication Inhibitor.

A high-throughput screen of a Roche internal chemical library based on inhibition of the respiratory syncytial virus (RSV)-induced cytopathic effect (CPE) on HEp-2 cells was performed to identify RSV inhibitors. Over 2,000 hits were identified and confirmed to be efficacious against RSV infection in vitro Here, we report the discovery of a triazole-oxadiazole derivative, designated triazole-1, as an RSV replication inhibitor, and we characterize its mechanism of action. Triazole-1 inhibited the replication of both RSV A and B subtypes with 50% inhibitory concentration (IC50) values of approximately 1 µM, but it was not effective against other viruses, including influenza virus A, human enterovirus 71 (EV71), and vaccinia virus. Triazole-1 was shown to inhibit RSV replication when added at up to 8 h after viral entry, suggesting that it inhibits RSV after viral entry. In a minigenome reporter assay in which RSV transcription regulatory sequences flanking a luciferase gene were cotransfected with RSV N/P/L/M2-1 genes into HEp-2 cells, triazole-1 demonstrated specific and dose-dependent RSV transcription inhibitory effects. Consistent with these findings, deep sequencing of the genomes of triazole-1-resistant mutants revealed a single point mutation (A to G) at nucleotide 13546 of the RSV genome, leading to a T-to-A change at amino acid position 1684 of the L protein, which is the RSV RNA polymerase for both viral transcription and replication. The effect of triazole-1 on minigenome transcription, which was mediated by the L protein containing the T1684A mutation, was significantly reduced, suggesting that the T1684A mutation alone conferred viral resistance to triazole-1.

HBVcircle: A novel tool to investigate hepatitis B virus covalently closed circular DNA.

BACKGROUND & AIMS: Hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) persists as a stable episome in infected hepatocytes and serves as a template for the transcription of all viral genes. Due to the narrow host range of HBV, the development of a robust mouse model that supports cccDNA-dependent viral replication is a key hurdle in the development of novel HBV therapeutics. This study aimed to develop a novel tool to investigate HBV cccDNA. METHODS: Through minicircle technology, HBVcircle, a recombinant cccDNA, was easily generated and extracted from a genetically engineered E. coli strain. We characterized the performance of HBVcircle in cell culture by transfection and in immunocompetent mice by hydrodynamic injection (HDI). RESULTS: We demonstrated that HBVcircle formed authentic cccDNA-like molecules in vitro in transiently transfected hepatic cells and in vivo in mouse liver after HDI. HBVcircle supported high levels and persistent HBV replication. In addition, we investigated different factors affecting HBV in vivo replication and persistence, including the host genetic background, vector design and dosage, viral genes and genotypes, and immune activation status. Furthermore, different classes of anti-HBV drugs were also assessed with the HBVcircle system. CONCLUSION: Compared with previous reported HBV mouse models which employ other viral vectors to introduce overlength HBV genomes, viral gene expression and associated phenotypes are entirely driven by cccDNA-like viral genomes in the HBVcircle mouse model. Therefore, the HBVcircle is a close mimic of cccDNA, and it represents a novel tool for addressing HBV cccDNA related biological questions and for anti-HBV drug discovery. LAY SUMMARY: To establish a mouse model that supports cccDNA-dependent transcription, a novel tool named HBVcircle, was developed with minicircle technology. HBVcircle formed authentic cccDNA-like molecules in hepatocytes, and supported high levels and persistent HBV replication in vivo. The HBVcircle is a close mimic of cccDNA, and it represents a novel tool for addressing HBV cccDNA related biological questions and for anti-HBV drug discovery.

Impact of Preseason Climate Factors on Vegetation Photosynthetic Phenology in Mid-High Latitudes of the Northern Hemisphere.

During the period preceding the vegetation growing season (GS), temperature emerges as the pivotal factor determining phenology in northern terrestrial ecosystems. Despite extensive research on the impact of daily mean temperature (Tmean) during the preseason period, the influence of diurnal temperature range (DTR) on vegetation photosynthetic phenology (i.e., the impact of the plant photosynthetic cycle on seasonal time scale) has largely been neglected. Using a long-term vegetation photosynthetic phenology dataset and historical climate data, we examine vegetation photosynthetic phenology dynamics and responses to climate change across the mid-high latitudes of the Northern Hemisphere from 2001 to 2020. Our data reveal an advancing trend in the start of the GS (SOS) by -0.15 days per year (days yr-1), affecting 72.1% of the studied area. This is particularly pronounced in western Canada, Alaska, eastern Asia, and latitudes north of 60°N. Conversely, the end of the GS (EOS) displays a delaying trend of 0.17 days yr-1, impacting 62.4% of the studied area, especially northern North America and northern Eurasia. The collective influence of an earlier SOS and a delayed EOS has resulted in the notably prolonged length of the GS (LOS) by 0.32 days yr-1 in the last two decades, affecting 70.9% of the studied area, with Eurasia and western North America being particularly noteworthy. Partial correlation coefficients of the SOS with preseason Tmean, DTR, and accumulated precipitation exhibited negative values in 98.4%, 93.0%, and 39.2% of the study area, respectively. However, there were distinct regional variations in the influence of climate factors on the EOS. The partial correlation coefficients of the EOS with preseason Tmean, DTR, and precipitation were positive in 58.6%, 50.1%, and 36.3% of the region, respectively. Our findings unveil the intricate mechanisms influencing vegetation photosynthetic phenology, holding crucial significance in understanding the dynamics of carbon sequestration within terrestrial ecosystems amidst climate change.

The Seasonal Divergence in the Weakening Relationship between Interannual Temperature Changes and Northern Boreal Vegetation Activity.

The response of boreal vegetation to global warming has shown a weakening trend over the last three decades. However, in previous studies, models of vegetation activity responses to temperature change have often only considered changes in the mean daily temperature (Tmean), with the diurnal temperature range (DTR) being neglected. The goal of this study was to evaluate the temporal trends of the relationships between two temperature factors (Tmean and DTR) and the vegetation activity across the boreal regions on both annual and seasonal timescales, by simultaneously employing satellite and climate datasets. We found that the interannual partial correlation between the growing season (GS) NDVI and Tmean (RNDVI-Tmean) has shown a significant decreasing trend over the last 34 years. At the seasonal scale, the RNDVI-Tmean showed a significant upward trend in the spring, while in the summer and autumn, the RNDVI-Tmean exhibited a significant downward trend. The temporal trend characteristics of the partial correlation between the NDVI and DTR (RNDVI-DTR), at both the GS and seasonal scales, were fully consistent with the RNDVI-Tmean. The area with a significant decrease in the GS RNDVI-Tmean and RNDVI-DTR accounted for approximately 44.4% and 41.2% of the boreal region with the 17-year moving window, respectively. In stark contrast, the area exhibiting a significant increasing trend in the GS RNDVI-Tmean and RNDVI-DTR accounted for only approximately 22.3% and 25.8% of the boreal region with the 17-year moving window, respectively. With respect to the seasonal patterns of the RNDVI-Tmean and RNDVI-DTR, the area with a significant upward trend in the spring was greater than that with a significant downward trend. Nevertheless, more areas had a significant downward trend in the RNDVI-Tmean and RNDVI-DTR in summer and autumn than a significant upward trend. Overall, our research reveals a weakening trend in the impact of temperature on the vegetation activity in the boreal regions and contributes to a deeper understanding of the vegetation response to global warming.

Varying Responses of Vegetation Greenness to the Diurnal Warming across the Global.

The distribution of global warming has been varying both diurnally and seasonally. Little is known about the spatiotemporal variations in the relationships between vegetation greenness and day- and night-time warming during the last decades. We investigated the global inter- and intra-annual responses of vegetation greenness to the diurnal asymmetric warming during the period of 1982-2015, using the normalized different vegetation index (NDVI, a robust proxy for vegetation greenness) obtained from the NOAA/AVHRR NDVI GIMMS3g dataset and the monthly average daily maximum (Tmax) and minimum temperature (Tmin) obtained from the gridded Climate Research Unit, University of East Anglia. Several findings were obtained: (1) The strength of the relationship between vegetation greenness and the diurnal temperature varied on inter-annual and seasonal timescales, indicating generally weakening warming effects on the vegetation activity across the global. (2) The decline in vegetation response to Tmax occurred mainly in the mid-latitudes of the world and in the high latitudes of the northern hemisphere, whereas the decline in the vegetation response to Tmin primarily concentrated in low latitudes. The percentage of areas with a significantly negative trend in the partial correlation coefficient between vegetation greenness and diurnal temperature was greater than that of the areas showing the significant positive trend. (3) The trends in the correlation between vegetation greenness and diurnal warming showed a complex spatial pattern: the majority of the study areas had undergone a significant declining strength in the vegetation greenness response to Tmax in all seasons and to Tmin in seasons except autumn. These findings are expected to have important implications for studying the diurnal asymmetry warming and its effect on the terrestrial ecosystem.

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.

Discovery of Ziresovir as a Potent, Selective, and Orally Bioavailable Respiratory Syncytial Virus Fusion Protein Inhibitor.

Ziresovir (RO-0529, AK0529) is reported here for the first time as a promising respiratory syncytial virus (RSV) fusion (F) protein inhibitor that currently is in phase 2 clinical trials. This article describes the process of RO-0529 as a potent, selective, and orally bioavailable RSV F protein inhibitor and highlights the in vitro and in vivo anti-RSV activities and pharmacokinetics in animal species. RO-0529 demonstrates single-digit nM EC50 potency against laboratory strains, as well as clinical isolates of RSV in cellular assays, and more than one log viral load reduction in BALB/c mouse model of RSV viral infection. RO-0529 was proven to be a specific RSV F protein inhibitor by identification of drug resistant mutations of D486N, D489V, and D489Y in RSV F protein and the inhibition of RSV F protein-induced cell-cell fusion in cellular assays.

Molecular epidemiology of human immunodeficiency virus type 1 and hepatitis C virus in former blood donors in central China.

The prevalence of hepatitis C virus (HCV) among human immunodeficiency virus (HIV-1)-positive former blood donors (FBDs) in Hubei province, central China, and the subtypes of these two viruses are identified. HIV-1-positive specimens were collected from FBDs, transfusion recipients, and their sexual partners in Hubei province, central China. The prevalence of HCV in HIV-1-positive FBDs was 78.6%. The dominant circulating HIV-1 subtype of FBDs was subtype B' (Thai-B); one belonged to U.S.-European subtype B. HCV genotypes 2a (78.6%) and 1b (21.4%) were detected. No recombinant form of HIV-1 was identified. Non-B' subtypes occurring among FBDs indicate the complexity of the HIV-1 prevalence in central China, where HIV is beginning to spread into the general population.

Role of the PI3K­AKT­mTOR pathway in hepatitis B virus infection and replication.

The replication of hepatitis B virus (HBV) may be modulated by a variety of cell signaling pathways, including the phosphatidylinositol 3­kinase (PI3K)­RAC­α serine/threonine­protein kinase (AKT)­serine/threonine­protein kinase mTOR (mTOR) pathway. The aim of the present study was to determine the regulatory effects of this pathway on the infection and replication of HBV. The results indicated that the HBV entry process may activate the AKT pathway, as demonstrated by AKT phosphorylation in HBV natural infection. However, inhibition of AKT phosphorylation by short­term treatment with AKT inhibitors was unable to block HBV entry, which suggested that AKT activation induced by HBV infection is not essential for viral entry process. Prolonged treatment with PI3K­AKT­mTOR pathway inhibitors markedly promoted HBV replication in HBV replicating and natural infection models. The PI3K­AKT­mTOR pathway was therefore identified to be a negative regulator of HBV replication. These inhibitors enhanced the replication and transcription of HBV in an HBx­dependent way. The results additionally indicated that a PI3K inhibitor, Ly294002, inhibited the secretion of the small surface antigen of HBV in a PI3K­AKT­independent manner. The inhibitor Ly294002 may be used as a tool for the drug development of surface antigen secretion inhibitors.

Discovery of Piperazinylquinoline Derivatives as Novel Respiratory Syncytial Virus Fusion Inhibitors.

A novel series of piperazinylquinoline derivatives were discovered as respiratory syncytial virus (RSV) fusion inhibitors by the ligand-based screening approach. Among 3,000 hits, 1-amino-3-[[2-(4-phenyl-1-piperidyl)-4-quinolyl]amino]propan-2-ol (7) was proven to be active against the RSV long (A) strain. The anti-RSV activity was improved by converting piperidine to benzylcarbonyl substituted piperazine. The basic side chain was also found to be crucial for anti-RSV activity. The selected analogues, 45 and 50, demonstrated anti-RSV activities up to EC50 = 0.028 µM and 0.033 µM, respectively. A direct anti-RSV effect was confirmed by a plaque reduction assay and a fusion inhibition assay. Both 45 and 50 showed promising DMPK properties with good oral bioavailability, and could potentially lead to novel therapeutic agents targeting the RSV fusion process.