Hui-Chen Hung

Aurintricarboxylic acid inhibits influenza virus neuraminidase

Abstract There is a continuing threat that the highly pathogenic avian influenza virus will cause future influenza pandemics. In this study, we screened a library of compounds that are biologically active and structurally diverse for inhibitory activity against influenza neuraminidase (NA). We found that aurintricarboxylic acid (ATA) is a potent inhibitor of NA activity of both group-1 and group-2 influenza viruses with IC50s (effective concentration to inhibit NA activity by 50%) values at low micromolar concentrations. ATA was equally potent in inhibiting the NA activity derived from wild-type NA and its H274Y mutant which renders NA resistance to inhibition by oseltamivir. Although ATA is structurally distinct from sialic acid, molecular modeling experiments suggested that ATA binds to NA at the enzyme’s substrate binding site. These results indicate that ATA may be a good starting material for the design of a novel class of NA inhibitors for the treatment influenza viruses.

Synergistic Inhibition of Enterovirus 71 Replication by Interferon and Rupintrivir

BACKGROUND Enterovirus 71 (EV71) can cause severe diseases and even lead to death in children. There is no vaccine or specific antiviral therapy to prevent or cure EV71 infection. Although interferon (IFN)-α has been used in the treatment of several viral infections, we found that IFN-α alone was ineffective in restricting EV71 replication in Vero cells. METHODS Through a bioinformatics analysis, several cellular proteins in the IFN response pathway were identified as susceptible substrates that might be degraded by the EV71-encoded 3C protease (3C(pro)). RESULTS Indeed, IRF9 was shown to be vulnerable to 3C(pro) cleavage, as revealed by enzyme-based and cell-based assays. Thus, the IFN-mediated antiviral mechanism compromised by the viral 3C(pro) in EV71-infected cells may be accountable, at least partially, for that IFN-α cannot inhibit EV71 replication. Because rupintrivir (AG7088) is known to be an effective EV71 inhibitor, we investigated the effects of the combination of rupintrivir and IFN-α on EV71 replication and found that they strongly synergized with each other in inhibiting EV71 replication. CONCLUSIONS Because rupintrivir was shown to be generally tolerable in earlier clinical investigations, it is worth evaluating whether a combination of rupintrivir and IFN-α could be an effective treatment for EV71.

Inhibition of enterovirus 71 replication and the viral 3D polymerase by aurintricarboxylic acid

Abstract Objectives Enterovirus 71 (EV71) causes serious diseases in humans. The aim of this study was to examine the effects of aurintricarboxylic acid (ATA) on EV71 replication and to explore the underlying mechanism. Methods To measure the activity of ATA in inhibiting the cytopathic effect (CPE) of EV71, a cell-based neutralization (inhibition of virus-induced CPE) assay was performed. The effect of ATA was further confirmed using plaque reduction and viral yield reduction assays. A time of addition assay was performed to identify the mechanisms of ATAs anti-EV71 activity. We examined the effects of ATA on the following key steps involved in virus replication: (i) translation of the internal ribosomal entry site (IRES)-mediated viral polyprotein; (ii) the proteolytic activity of viral proteases 2A and/or 3C; and (iii) the viral 3D RNA-dependent RNA polymerase (RdRp) activity. Results In this study, ATA was found to be a potent inhibitor of the replication of EV71. In the antiviral neutralization assay, ATA exhibited inhibitory activity against EV71 (TW/4643/98) and EV71 (TW/2231/98). Plaque assay further demonstrated that ATA inhibited EV71 replication with an EC50 (effective concentration at which 50% of plaques were removed) of 2.9 µM. Studies on the mechanism of action revealed that ATA targets the early stage of the viral life cycle after viral entry. ATA was able to inhibit the RdRp activity of EV71, while neither the IRES-mediated translation of viral polyprotein nor the viral 3C protease activity was affected. Conclusions Overall, the findings in this study suggest that ATA is able to effectively inhibit EV71 replication through interfering with the viral 3D polymerase.

Identification of BPR3P0128 as an Inhibitor of Cap-Snatching Activities of Influenza Virus

ABSTRACT The aim of this study was to identify the antiviral mechanism of a novel compound, BPR3P0128. From a large-scale screening of a library of small compounds, BPR3P compounds were found to be potent inhibitors of influenza viral replication in Madin–Darby canine kidney (MDCK) cells. BPR3P0128 exhibited inhibitory activity against both influenza A and B viruses. The 50% inhibitory concentrations were in the range of 51 to 190 nM in MDCK cells, as measured by inhibition-of-cytopathic-effect assays. BPR3P0128 appeared to target the viral replication cycle but had no effect on viral adsorption. The inhibition of cap-dependent mRNA transcription by BPR3P0128 was more prominent with a concurrent increase in cap-independent cRNA replication in a primer extension assay, suggesting a role of BPR3P0128 in switching transcription to replication. This reduction in mRNA expression resulted from the BPR3P-mediated inhibition of the cap-dependent endoribonuclease (cap-snatching) activities of nuclear extracts containing the influenza virus polymerase complex. No inhibition of binding of 5′ viral RNA to the viral polymerase complex by this compound was detected. BPR3P0128 also effectively inhibited other RNA viruses, such as enterovirus 71 and human rhinovirus, but not DNA viruses, suggesting that BPR3P0128 targets a cellular factor(s) associated with viral PB2 cap-snatching activity. The identification of this factor(s) could help redefine the regulation of viral transcription and replication and thereby provide a potential target for antiviral chemotherapeutics.

Development of an Anti-Influenza Drug Screening Assay Targeting Nucleoproteins with Tryptophan Fluorescence Quenching

Recent studies have shown that NP (nucleoprotein), which possesses multiple functions in the viral life cycle, is a new potential anti-influenza drug target. NP inhibitors reliably induce conformational changes in NPs, and these changes may confer inhibition of the influenza virus. The six conserved tryptophan residues in NP can be used as an intrinsic probe to monitor the change in fluorescence of the tryptophan residues in the protein upon binding to an NP inhibitor. In the present study, we found that the fluorescence of recombinant NP proteins was quenched following the binding of available NP inhibitors (such as nucleozin) in a concentration- and time-dependent manner, which suggests that the inhibitor induced conformational changes in the NPs. The minimal fluorescence-quenching effect and weak binding constant of nucleozin to the swine-origin influenza virus H1N1pdm09 (SOIV) NP revealed that the SOIV is resistant to nucleozin. We have used the fluorescence-quenching property of tryptophans in NPs that were bound to ligands in a 96-well-plate-based drug screen to assess the ability of promising small molecules to interact with NPs and have identified one new anti-influenza drug, CSV0C001018, with a high SI value. This convenient method for drug screening may facilitate the development of antiviral drugs that target viruses other than the influenza virus, such as HIV and HBV.

Parallel Screening of Wild-Type and Drug-Resistant Targets for Anti-Resistance Neuraminidase Inhibitors

Infection with influenza virus is a major public health problem, causing serious illness and death each year. Emergence of drug-resistant influenza virus strains limits the effectiveness of drug treatment. Importantly, a dual H275Y/I223R mutation detected in the pandemic influenza A 2009 virus strain results in multidrug resistance to current neuraminidase (NA) drugs. Therefore, discovery of new agents for treating multiple drug-resistant (MDR) influenza virus infections is important. Here, we propose a parallel screening strategy that simultaneously screens wild-type (WT) and MDR NAs, and identifies inhibitors matching the subsite characteristics of both NA-binding sites. These may maintain their potency when drug-resistant mutations arise. Initially, we analyzed the subsite of the dual H275Y/I223R NA mutant. Analysis of the site-moiety maps of NA protein structures show that the mutant subsite has a relatively small volume and is highly polar compared with the WT subsite. Moreover, the mutant subsite has a high preference for forming hydrogen-bonding interactions with polar moieties. These changes may drive multidrug resistance. Using this strategy, we identified a new inhibitor, Remazol Brilliant Blue R (RB19, an anthraquinone dye), which inhibited WT NA and MDR NA with IC50 values of 3.4 and 4.5 µM, respectively. RB19 comprises a rigid core scaffold and a flexible chain with a large polar moiety. The former interacts with highly conserved residues, decreasing the probability of resistance. The latter forms van der Waals contacts with the WT subsite and yields hydrogen bonds with the mutant subsite by switching the orientation of its flexible side chain. Both scaffolds of RB19 are good starting points for lead optimization. The results reveal a parallel screening strategy for identifying resistance mechanisms and discovering anti-resistance neuraminidase inhibitors. We believe that this strategy may be applied to other diseases with high mutation rates, such as cancer and human immunodeficiency virus type 1.

Using mutagenesis to explore conserved residues in the RNA-binding groove of influenza A virus nucleoprotein for antiviral drug development

Nucleoprotein (NP) is the most abundant type of RNA-binding viral protein in influenza A virus–infected cells and is necessary for viral RNA transcription and replication. Recent studies demonstrated that influenza NP is a valid target for antiviral drug development. The surface of the groove, covered with numerous conserved residues between the head and body domains of influenza A NP, plays a crucial role in RNA binding. To explore the mechanism by which NP binds RNA, we performed a series of site-directed mutagenesis in the RNA-binding groove, followed by surface plasmon resonance (SPR), to characterize the interactions between RNA and NP. Furthermore, a role of Y148 in NP stability and NP-RNA binding was evaluated. The aromatic residue of Y148 was found to stack with a nucleotide base. By interrupting the stacking interaction between Y148 and an RNA base, we identified an influenza virus NP inhibitor, (E, E)-1,7-bis(4-hydroxy-3-methoxyphenyl) -1,6-heptadiene-3,5-dione; this inhibitor reduced the NP’s RNA-binding affinity and hindered viral replication. Our findings will be useful for the development of new drugs that disrupt the interaction between RNA and viral NP in the influenza virus.

The Combination Effects of LiCl and the Active Leflunomide Metabolite, A771726, on Viral-Induced Interleukin 6 Production and EV-A71 Replication

Enterovirus 71 (EV-A71) is a neurotropic virus that can cause severe complications involving the central nervous system. No effective antiviral therapeutics are available for treating EV-A71 infection and drug discovery efforts are rarely focused to target this disease. Thus, the main goal of this study was to discover existing drugs with novel indications that may effectively inhibit EV-A71 replication and the inflammatory cytokines elevation. In this study, we showed that LiCl, a GSK3β inhibitor, effectively suppressed EV-A71 replication, apoptosis and inflammatory cytokines production (Interleukin 6, Interleukin-1β) in infected cells. Furthermore, LiCl and an immunomodular agent were shown to strongly synergize with each other in suppressing EV-A71 replication. The results highlighted potential new treatment regimens in suppressing sequelae caused by EV-A71 replication.

Anti-influenza Drug Discovery: Identification of an Orally Bioavailable Quinoline Derivative through Activity- and Property-Guided Lead Optimization

From a high-throughput screening (HTS) hit with inhibitory activity against virus-induced cytophathic in the low micromolar range, we have developed a potent anti-influenza lead through careful optimization without compromising the drug-like properties of the compound. An orally bioavailable compound was identified as a lead agent with nanomolar activity against influenza, representing a 140-fold improvement over the initial hit.

Identification of neuraminidase inhibitors against dual H274Y/I222R mutant strains

Influenza is an annual seasonal epidemic that has continually drawn public attentions, due to the potential death toll and drug resistance. Neuraminidase, which is essential for the spread of influenza virus, has been regarded as a valid target for the treatment of influenza infection. Although neuraminidase drugs have been developed, they are susceptible to drug-resistant mutations in the sialic-binding site. In this study, we established computational models (site-moiety maps) of H1N1 and H5N1 to determine properties of the 150-cavity, which is adjacent to the drug-binding site. The models reveal that hydrogen-bonding interactions with residues R118, D151, and R156 and van der Waals interactions with residues Q136, D151, and T439 are important for identifying 150-cavitiy inhibitors. Based on the models, we discovered three new inhibitors with IC50 values <10 μM that occupies both the 150-cavity and sialic sites. The experimental results identified inhibitors with similar activities against both wild-type and dual H274Y/I222R mutant neuraminidases and showed little cytotoxic effects. Furthermore, we identified three new inhibitors situated at the sialic-binding site with inhibitory effects for normal neuraminidase, but lowered effects for mutant strains. The results suggest that the new inhibitors can be used as a starting point to combat drug-resistant strains.