Preclinical characterization of AB-506, an inhibitor of HBV replication targeting the viral core protein.

AB-506, a small-molecule inhibitor targeting the HBV core protein, inhibits viral replication in vitro (HepAD38 cells: EC50 of 0.077 µM, CC50 > 25 µM) and in vivo (HBV mouse model: ∼3.0 log10 reductions in serum HBV DNA compared to the vehicle control). Binding of AB-506 to HBV core protein accelerates capsid assembly and inhibits HBV pgRNA encapsidation. Furthermore, AB-506 blocks cccDNA establishment in HBV-infected HepG2-hNTCP-C4 cells and primary human hepatocytes, leading to inhibition of viral RNA, HBsAg, and HBeAg production (EC50 from 0.64 µM to 1.92 µM). AB-506 demonstrated activity across HBV genotypes A-H and maintains antiviral activity against nucleos(t)ide analog-resistant variants in vitro. Evaluation of AB-506 against a panel of core variants showed that T33N/Q substitutions results in >200-fold increase in EC50 values, while L30F, L37Q, and I105T substitutions showed an 8 to 20-fold increase in EC50 values in comparison to the wild-type. In vitro combinations of AB-506 with NAs or an RNAi agent were additive to moderately synergistic. AB-506 exhibits good oral bioavailability, systemic exposure, and higher liver to plasma ratios in rodents, a pharmacokinetic profile supporting clinical development for chronic hepatitis B.

Discovery of Novel Pyrimidine-Based Capsid Assembly Modulators as Potent Anti-HBV Agents.

Core assembly modulators of viral capsid proteins have been developed as an effective treatment of chronic hepatitis B virus (HBV) infection. In this study, we synthesized novel potent pyrimidine derivatives as core assembly modulators, and their antiviral effects were evaluated in in vitro and in vivo biological experiments. One of the synthesized derivatives, compound 23h (R1 = MeSO2, R2 = 1-piperidin-4-amine, R3 = 3-Cl-4-F-aniline) displayed potent inhibitory effects in the in vitro assays (52% inhibition in the protein-based assay at 100 nM and an IC50 value of 181 nM in the serum HBV DNA quantification assay). Moreover, treatment with compound 23h for 5 weeks significantly decreased serum levels of HBV DNA levels (3.35 log reduction) in a human liver-chimeric uPA/SCID mouse model, and these effects were significantly increased when 23h was combined with tenofovir, a nucleotide analogue inhibitor of reverse transcriptase used for the treatment of HBV infection.

An Antiviral Drug Screening Platform with a FRET Biosensor for Measurement of Arenavirus Z Assembly.

The smallest arenavirus gene product, Z protein, plays critical roles in the virus life cycle. Z is the major driving force of budding and particle production because of a unique property that defines self-assembly. In addition to the roles in budding, Z also participates in the suppression of type I interferon production to evade host antiviral immunity. Therefore, Z and its assembled form are an attractive drug target for arenaviral hemorrhagic fever, such as Lassa fever. Here, we developed a biosensor that enabled the evaluation of the prototype arenavirus, lymphocytic choriomeningitis virus (LCMV), Z assembly using the principle of Förster resonance energy transfer (FRET). This FRET biosensor consisted of three tandem Z that were sandwiched between super-enhanced cyan-emitting fluorescent protein and variant of a yellow-emitting mutant of green fluorescent protein so that Z-Z intermolecular binding via the really interesting new gene finger domain increased the emission ratio. To identify novel anti-arenavirus compounds, the FRET biosensor was employed to screen the PathogenBox400 for inhibitors of Z assembly in a 96-well plate format. The assay performed well, with a Z'-factor of 0.89, and identified two compounds that decreased the emission ratio of the FRET biosensor in a dose-dependent manner. Of them, the compound, 5,6,7,8-tetrahydro-7-(benzyl)-pyrido[4',3':4,5]thieno[2,3-d]pyrimidin-2,4-diamine, was found to significantly inhibit LCMV propagation in infected cells. Thereby, the present study demonstrated that a novel FRET biosensor incorporating Z assembly built on FRET and named Zabton, was a valuable screening tool to identify anti-arenavirus compounds in the context of inhibition of Z assembly.Key words: Arenavirus, Förster resonance energy transfer, anti-viral drugs, Z protein.

Pyrimidotriazine derivatives as selective inhibitors of HBV capsid assembly.

Chronic hepatitis B virus (HBV) infection is currently treated with nucleoside/nucleotides analogs. They are potent inhibitors of HBV DNA polymerase, which also functions as reverse transcriptase. Although nucleoside/nucleotide analogs efficiently suppress HBV replication in liver cells, they cannot eradicate HBV DNA from liver cells and cure the disease. Therefore, it is still mandatory to identify and develop effective inhibitors that target a step other than reverse transcription in the viral replication cycle. HBV capsid assembly is a critical step for viral replication and an attractive target for inhibition of HBV replication. We conducted in silico screening of compounds expected to bind to the HBV capsid dimer-dimer interaction site. The selected compounds were further examined for their anti-HBV activity in vitro. Among the test compounds, novel pyrimidotriazine derivatives were found to be selective inhibitors of HBV replication in HepG2.2.15.7 cells. Among the compounds, 2-[(2,3-dichlorophenyl)amino]-4-(4-tert-butylphenyl)-8-methyl-4H,9H-pyrimido[1,2-a][1,3,5]triazin-6-one was the most active against HBV replication. Studies on its mechanism of action revealed that the compound interfered with HBV capsid assembly determined by a cell-free capsid assembly system. Thus, the pyrimidotriazine derivatives are considered to be potential leads for novel HBV capsid assembly inhibitors.

A HTRF based competitive binding assay for screening specific inhibitors of HIV-1 capsid assembly targeting the C-Terminal domain of capsid.

Due to its multifaceted essential roles in virus replication and extreme genetic fragility, the human immunodeficiency virus type 1 (HIV-1) capsid (CA) protein is a valued therapeutic target. However, CA is as yet unexploited clinically, as there are no antiviral agents that target it currently on the market. To facilitate the identification of potential HIV-1 CA inhibitors, we established a homogeneous time-resolved fluorescence (HTRF) assay to screen for small molecules that target a biologically active and specific binding pocket in the C-terminal domain of HIV-1 CA (CA CTD). The assay, which is based on competition of small molecules for the binding of a known CA inhibitor (CAI) to the CA CTD, exhibited a signal-to-background ratio (S/B) > 10 and a Z' value > 0.9. In a pilot screen of three kinase inhibitor libraries containing 464 compounds, we identified one compound, TX-1918, as a low micromolecular inhibitor of the HIV-1 CA CTD-CAI interaction (IC50 = 3.81 µM) that also inhibited viral replication at moderate micromolar concentration (EC50 = 15.16 µM) and inhibited CA assembly in vitro. Based on the structure of TX-1918, an additional compound with an antiviral EC50 of 6.57 µM and cellular cytotoxicity CC50 of 102.55 µM was obtained from a compound similarity search. Thus, the HTRF-based assay has properties that are suitable for screening large compound libraries to identify novel anti-HIV-1 inhibitors targeting the CA CTD.

Ciclopirox inhibits Hepatitis B Virus secretion by blocking capsid assembly.

Chronic hepatitis B virus (HBV) infection can cause cirrhosis and hepatocellular carcinoma and is therefore a serious public health problem. Infected patients are currently treated with nucleoside/nucleotide analogs and interferon α, but this approach is not curative. Here, we screen 978 FDA-approved compounds for their ability to inhibit HBV replication in HBV-expressing HepG2.2.15 cells. We find that ciclopirox, a synthetic antifungal agent, strongly inhibits HBV replication in cells and in mice by blocking HBV capsid assembly. The crystal structure of the HBV core protein and ciclopirox complex reveals a unique binding mode at dimer-dimer interfaces. Ciclopirox synergizes with nucleoside/nucleotide analogs to prevent HBV replication in cells and in a humanized liver mouse model. Therefore, orally-administered ciclopirox may provide a novel opportunity to combat chronic HBV infection by blocking HBV capsid assembly.

Design and synthesis of aminothiazole based Hepatitis B Virus (HBV) capsid inhibitors.

The capsid assembly is an essential step for Hepatitis B Virus (HBV) life cycle and is an important target for anti-HBV drug development. In this report, we identified a hit compound with aminothiazole structure by the high throughput screening (HTS) which inhibited the interaction of HBV capsid protein within the cells. The structure hopping and SAR studies of the hit compound afforded compound 79 with potent anti-HBV replication activity and good basic drug-like properties. The working mechanism studies showed that compound 79 could bind to the similar binding site of known HBV capsid inhibitor with heteroaryldihydropyrimidine (HAP) scaffold, through similar hydrophobic interactions but with a different hydrogen bond. This compound exerted potent inhibitory effect upon HBV production, either in cell culture or in mice with no obvious acute toxicity. We propose that further development of this compound could lead to novel potent anti-HBV inhibitors that target HBV capsid assembly.

Luteolin escape mutants of dengue virus map to prM and NS2B and reveal viral plasticity during maturation.

We previously showed that luteolin, a well-known plant-derived component found in the "heat clearing" class of Traditional Chinese Medicine (TCM) herbs, is an uncompetitive inhibitor (Ki 58.6 µM) of the host proprotein convertase furin, an endoprotease that is required for maturation of flaviviruses in the trans-Golgi compartment. Luteolin also weakly inhibited recombinant dengue virus NS2B/NS3 protease (Ki 140.36 µM) non-competitively. In order to further explore the mechanism of inhibition we isolated resistant mutants by continuous passaging of DENV2 in the presence of increasing concentrations of luteolin. Nucleotide sequence analysis of the luteolin-resistant escape mutants revealed nucleotide changes that lead to amino acid substitutions in the prM (T79R) and NS2B (I114M) genes. These mutations were introduced into a DENV2 infectious clone and tested for replication in Huh-7 cells. Interestingly we found that the replication kinetics of prM T19R-NS2B I114M double-mutant (DM) was similar to wild-type virus (WT). On the other hand the prM T79R single mutant (SM1) was attenuated and the NS2B I114M single mutant (SM2) showed enhanced replication. Time of drug addition assay with luteolin showed that the mutant viruses were able to produce more mature virions than WT in the order DM > SM2>SM1>WT. Exogenous addition of furin to purified immature WT or mutant viruses revealed that luteolin blocked the prM cleavage of WT and SM2 at a similar level. On the other hand the SM1 immature virus showed some cleavage while the DM immature virus revealed efficient furin cleavage of prM even in the presence of 50 µM luteolin. Our findings suggest that luteolin inhibition of furin may occur at host/pathogen interface that permits the virus to escape the suppression by mutating key residue that may lead to an altered interface.

Plant-derived antivirals against hepatitis c virus infection.

Hepatitis C virus (HCV) infection is a worldwide public health burden and it is estimated that 185 million people are or have previously been infected worldwide. There is no effective vaccine for prevention of HCV infection; however, a number of drugs are available for the treatment of infection. The availability of direct-acting antivirals (DAAs) has dramatically improved therapeutic options for HCV genotype 1. However, the high costs and potential for development of resistance presented by existing treatment demonstrate the need for the development of more efficient new antivirals, or combination of therapies that target different stages of the viral lifecycle. Over the past decades, there has been substantial study of compounds extracted from plants that have activity against a range of microorganisms that cause human diseases. An extensive variety of natural compounds has demonstrated antiviral action worldwide, including anti-HCV activity. In this context, plant-derived compounds can provide an alternative approach to new antivirals. In this review, we aim to summarize the most promising plant-derived compounds described to have antiviral activity against HCV.

Discovery and Mechanistic Study of Benzamide Derivatives That Modulate Hepatitis B Virus Capsid Assembly.

Chronic hepatitis B virus (HBV) infection is a global public health problem. Although the currently approved medications can reliably reduce the viral load and prevent the progression of liver diseases, they fail to cure the viral infection. In an effort toward discovery of novel antiviral agents against HBV, a group of benzamide (BA) derivatives that significantly reduced the amount of cytoplasmic HBV DNA were discovered. The initial lead optimization efforts identified two BA derivatives with improved antiviral activity for further mechanistic studies. Interestingly, similar to our previously reported sulfamoylbenzamides (SBAs), the BAs promote the formation of empty capsids through specific interaction with HBV core protein but not other viral and host cellular components. Genetic evidence suggested that both SBAs and BAs inhibited HBV nucleocapsid assembly by binding to the heteroaryldihydropyrimidine (HAP) pocket between core protein dimer-dimer interfaces. However, unlike SBAs, BA compounds uniquely induced the formation of empty capsids that migrated more slowly in native agarose gel electrophoresis from A36V mutant than from the wild-type core protein. Moreover, we showed that the assembly of chimeric capsids from wild-type and drug-resistant core proteins was susceptible to multiple capsid assembly modulators. Hence, HBV core protein is a dominant antiviral target that may suppress the selection of drug-resistant viruses during core protein-targeting antiviral therapy. Our studies thus indicate that BAs are a chemically and mechanistically unique type of HBV capsid assembly modulators and warranted for further development as antiviral agents against HBV.IMPORTANCE HBV core protein plays essential roles in many steps of the viral replication cycle. In addition to packaging viral pregenomic RNA (pgRNA) and DNA polymerase complex into nucleocapsids for reverse transcriptional DNA replication to take place, the core protein dimers, existing in several different quaternary structures in infected hepatocytes, participate in and regulate HBV virion assembly, capsid uncoating, and covalently closed circular DNA (cccDNA) formation. It is anticipated that small molecular core protein assembly modulators may disrupt one or multiple steps of HBV replication, depending on their interaction with the distinct quaternary structures of core protein. The discovery of novel core protein-targeting antivirals, such as benzamide derivatives reported here, and investigation of their antiviral mechanism may lead to the identification of antiviral therapeutics for the cure of chronic hepatitis B.

Determinants of the VP1/2A junction cleavage by the 3C protease in foot-and-mouth disease virus-infected cells.

The foot-and-mouth disease virus (FMDV) capsid precursor, P1-2A, is cleaved by FMDV 3C protease to yield VP0, VP3, VP1 and 2A. Cleavage of the VP1/2A junction is the slowest. Serotype O FMDVs with uncleaved VP1-2A (having a K210E substitution in VP1; at position P2 in cleavage site) have been described previously and acquired a second site substitution (VP1 E83K) during virus rescue. Furthermore, introduction of the VP1 E83K substitution alone generated a second site change at the VP1/2A junction (2A L2P, position P2' in cleavage site). These virus adaptations have now been analysed using next-generation sequencing to determine sub-consensus level changes in the virus; this revealed other variants within the E83K mutant virus population that changed residue VP1 K210. The construction of serotype A viruses with a blocked VP1/2A cleavage site (containing K210E) has now been achieved. A collection of alternative amino acid substitutions was made at this site, and the properties of the mutant viruses were determined. Only the presence of a positively charged residue at position P2 in the cleavage site permitted efficient cleavage of the VP1/2A junction, consistent with analyses of diverse FMDV genome sequences. Interestingly, in contrast to the serotype O virus results, no second site mutations occurred within the VP1 coding region of serotype A viruses with the blocked VP1/2A cleavage site. However, some of these viruses acquired changes in the 2C protein that is involved in enterovirus morphogenesis. These results have implications for the testing of potential antiviral agents targeting the FMDV 3C protease.

Ethyl 4-(4-methylphenyl)-4-pentenoate from Vetiveria zizanioides Inhibits Dengue NS2B-NS3 Protease and Prevents Viral Assembly: A Computational Molecular Dynamics and Docking Study.

Around 50 % of the world's population is at the risk of dengue, a viral infection. Presently, there are not many drugs and prophylactic measures available to control dengue viral infection, and hence, there is an urgent need to develop effective antidengue compound from natural sources. In the current study, we explored the antiviral properties of the medicinal plant Vetiveria zizanioides against dengue virus. Initially, the antiviral properties of active compounds were examined using docking analysis along with reference ligand. The enzyme-ligand complex which showed higher binding affinity than the reference ligand was employed for subsequent analysis. The stability of the top scoring enzyme-ligand complex was further validated using molecular simulation studies. On the whole, the study reveals that the compound Ethyl 4-(4-methylphenyl)-4-pentenoate has an effective antiviral property, which can serve as a potential lead molecule in drug discovery process.

Activity-based and fraction-guided analysis of Phyllanthus urinaria identifies loliolide as a potent inhibitor of hepatitis C virus entry.

Without a vaccine, hepatitis C virus (HCV) remains a global medical and socio-economic burden, predisposing about 170 million carriers worldwide to end-stage liver diseases including cirrhosis and hepatocellular carcinoma. Although the recently developed direct-acting antivirals (DAAs) have revolutionized hepatitis C treatment, most of them are unsuitable for monotherapy due to risks of resistance, thus necessitating combination with interferon (IFN)-alpha, ribavirin, or additional DAAs. More importantly, the high cost associated with the DAAs restricts their accessibility to most parts of the world. Developing novel cost-effective anti-HCV therapeutics may help expand the scope of antivirals and treatment strategies against hepatitis C. Herein, we applied an activity-based and fraction-guided analysis of extracts from the medicinal plant Phyllanthus urinaria (P. urinaria), which yielded fraction 13 (F13) as possessing the most potent inhibitory activity against early viral entry of cell-culture HCV infection. Chemical analysis (silica gel chromatography followed by ESI LC-MS plus (1)H and (13)C NMR) of F13 identified loliolide (LOD), a monoterpenoid lactone, as a novel inhibitor of HCV entry. Specifically, LOD could efficiently inactivate HCV free virus particles, abrogate viral attachment, and impede viral entry/fusion, with minimal effect on viral replication/translation, particle production, and induction of type I IFN host antiviral immune response. ELISA-based binding analysis confirmed the monoterpenoid's ability in efficiently blocking HCV particle attachment to the host cell surface. Furthermore, LOD could inhibit infection by several genotypic strains of HCV. This is the first report characterizing P. urinaria and its bioactive compound LOD as potent HCV entry inhibitors, which merit further evaluation for development as candidate antiviral agents against hepatitis C.

FAITH - Fast Assembly Inhibitor Test for HIV.

Due to the high number of drug-resistant HIV-1 mutants generated by highly active antiretroviral therapy (HAART), there is continuing demand for new types of inhibitors. Both the assembly of the Gag polyprotein into immature and mature HIV-1 particles are attractive candidates for the blocking of the retroviral life cycle. Currently, no therapeutically-used assembly inhibitor is available. One possible explanation is the lack of a reliable and simple assembly inhibitor screening method. To identify compounds potentially inhibiting the formation of both types of HIV-1 particles, we developed a new fluorescent high-throughput screening assay. This assay is based on the quantification of the assembly efficiency in vitro in a 96-well plate format. The key components of the assay are HIV-1 Gag-derived proteins and a dual-labelled oligonucleotide, which emits fluorescence only when the assembly of retroviral particles is inhibited. The method was validated using three (CAI, BM2, PF74) reported assembly inhibitors.

Vitamin D in liver diseases: from mechanisms to clinical trials.

Traditionally regarded as a typical vitamin regulating calcium and phosphorus homeostasis, vitamin D is now discovered as a highly versatile molecule with emerging roles in immunity, cancer, infectious diseases, fibrosis, fatty liver diseases, and alcoholic liver diseases. A large body of clinical evidence has demonstrated the prevalence and risks of vitamin D deficiency in various chronic diseases. Biologically active vitamin D, 1,25-dihydroxylvitamin D3, is synthesized in two distinct systems. In addition to the classic two-step hydroxylation in the liver and kidneys, 1,25-dihydroxylvitamin D3 can also be produced locally by immune cells in response to infection. The bioactive vitamin D generated in these two pools apparently functions differently: while the former facilitates calcium adsorption and homeostasis, the latter confers immune regulation. The immune regulatory functions of vitamin D are demonstrated by induction of antimicrobial peptides, suppression of innate immune response, induction of Th2 cytokines, and stimulation of T-regulatory T cells. Vitamin D deficiency or insufficiency is overwhelmingly associated with viral hepatitis, cirrhosis, and fatty liver diseases. Recent clinical trials have shown that vitamin D supplements significantly enhance the efficacy of interferon plus ribavirin therapy through sustained virological response. A recent study showed that 25-dihydroxyvitamin D rather than 1,25-dihydroxyvitamin D could directly suppress hepatitis C virus assembly. Moreover, clinical evidence has shown that vitamin D deficiency is associated with alcoholic and non-alcoholic fatty liver diseases. In this review, we highlight some recent advances in vitamin D researches and clinical trails.

Biotinylation of histones represses transposable elements in human and mouse cells and cell lines and in Drosophila melanogaster.

Transposable elements such as long terminal repeats (LTR) constitute approximately 45% of the human genome; transposition events impair genome stability. Fifty-four promoter-active retrotransposons have been identified in humans. Epigenetic mechanisms are important for transcriptional repression of retrotransposons, preventing transposition events, and abnormal regulation of genes. Here, we demonstrate that the covalent binding of the vitamin biotin to lysine-12 in histone H4 (H4K12bio) and lysine-9 in histone H2A (H2AK9bio), mediated by holocarboxylase synthetase (HCS), is an epigenetic mechanism to repress retrotransposon transcription in human and mouse cell lines and in primary cells from a human supplementation study. Abundance of H4K12bio and H2AK9bio at intact retrotransposons and a solitary LTR depended on biotin supply and HCS activity and was inversely linked with the abundance of LTR transcripts. Knockdown of HCS in Drosophila melanogaster enhances retrotransposition in the germline. Importantly, we demonstrated that depletion of H4K12bio and H2AK9bio in biotin-deficient cells correlates with increased production of viral particles and transposition events and ultimately decreases chromosomal stability. Collectively, this study reveals a novel diet-dependent epigenetic mechanism that could affect cancer risk.

Identification of novel antipoxviral agents: mitoxantrone inhibits vaccinia virus replication by blocking virion assembly.

The bioterror threat of a smallpox outbreak in an unvaccinated population has mobilized efforts to develop new antipoxviral agents. By screening a library of known drugs, we identified 13 compounds that inhibited vaccinia virus replication at noncytotoxic doses. The anticancer drug mitoxantrone is unique among the inhibitors identified in that it has no apparent impact on viral gene expression. Rather, it blocks processing of viral structural proteins and assembly of mature progeny virions. The isolation of mitoxantrone-resistant vaccinia strains underscores that a viral protein is the likely target of the drug. Whole-genome sequencing of mitoxantrone-resistant viruses pinpointed missense mutations in the N-terminal domain of vaccinia DNA ligase. Despite its favorable activity in cell culture, mitoxantrone administered intraperitoneally at the maximum tolerated dose failed to protect mice against a lethal intranasal infection with vaccinia virus.

Virtual screening and bioassay study of novel inhibitors for dengue virus mRNA cap (nucleoside-2'O)-methyltransferase.

We report high-throughput structure-based virtual screening of putative Flavivirus 2'-O-methyltransferase inhibitors together with results from subsequent bioassay tests of selected compounds. Potential inhibitors for the S-adenosylmethionine binding site were explored using 2D similarity searching, pharmacophore filtering and docking. The inhibitory activities of 15 top-ranking compounds from the docking calculations were tested on a recombinant methyltransferase with the RNA substrate (7Me)GpppAC(5). Local and global docking simulations were combined to estimate the ligand selectivity for the target site. The results of the combined computational and experimental screening identified a novel inhibitor, with a previously unknown scaffold, that has an IC(50) value of 60 microM.

Construction and characterization of a stable subgenomic dengue virus type 2 replicon system for antiviral compound and siRNA testing.

Self-replicating, non-infectious flavivirus subgenomic replicons have been broadly used in the studies of trans-complementation, adaptive mutation, viral assembly and packaging in Kunjin, yellow fever and West Nile viruses. We describe here the construction of subgenomic EGFP- or Renilla luciferase-reporter based dengue replicons of the type 2 New Guinea C (NGC) strain and the establishment of stable BHK21 cell lines harboring the replicons. In replicon cells, viral proteins and RNAs are stably expressed at levels similar to cells transfected with the full length NGC infectious RNA. Furthermore, the replicon can be packaged by separately transfected C (core)-prM (pre-membrane)-E (envelope) polyprotein construct. The replicon cells were subjected to treatment with several antiviral compounds and inhibition of the replicon was observed in treatment with known nucleoside analog inhibitors of NS5 such as 2'-C-methyladenosine (EC(50)=2.42 +/- 0.59 microM), or ribavirin (EC(50)=6.77 +/- 1.33 microM), mycophenolic acid (EC(50)=1.31 +/- 0.27 microM) and siRNA against NS3. The BHK-replicon cells have been stably maintained for about 10 passages without significant loss in reporter intensity and are sufficiently robust for both research and drug discovery.