Identification of quinone analogues as potential inhibitors of picornavirus 3C protease in vitro.

Picornaviruses are non-enveloped viruses that represent a large family of positive-sense single-stranded RNA viruses including a number of causative agents of many human and animal diseases such as coxsackievirus B3 (CVB3) and rhinoviruses (HRV). In this study, we performed a high-throughput screening of a compound library composed of ∼6000 small molecules in search of potential picornavirus 3C protease (3Cpro) inhibitors. As results, we identified quinone analogues that effectively inhibited both CVB3 3Cpro and HRV 3Cpro with IC50 values in low micromolar range. Together with predicted binding modes of these compounds to the active site of the viral protease, it is implied that structural features of these non-peptidic inhibitors may act as useful scaffold for further anti-picornavirus drug design and development.

Enterovirus inhibitory activity of C-8-tert-butyl substituted 4-aryl-6,7,8,9-tetrahydrobenzo[4,5]thieno[3,2-e][1,2,4]triazolo[4,3-a]pyrimidin-5(4H)-ones.

Members of a series of 4-aryl-6,7,8,9-tetrahydrobenzo[4,5]thieno[3,2-e][1,2,4]triazolo[4,3-a]pyrimidin-5(4H)-ones (1, Fig. 2) were prepared and tested against representative enteroviruses including Human Coxsackievirus B1 (Cox B1), Human Coxsackievirus B3 (Cox B3), human Poliovirus 3 (PV3), human Rhinovirus 14 (HRV14), human Rhinovirus 21 (HRV 21) and human Rhinovirus 71 (HRV 71). The C-8-tert-butyl group on the tetrahydrobenzene ring in these substances was found to be crucial for their enterovirus activity. One member of this group, 1e, showed single digit micromolar activities (1.6-8.85µM) against a spectrum of viruses screened, and the highest selectivity index (SI) values for Cox B1 (>11.2), for Cox B3 (>11.5), and for PV3 (>51.2), respectively. In contrast, 1p, was the most active analog against the selected HRVs (1.8-2.6µM), and showed the highest selectivity indices among the group of compounds tested. The SI values for 1p were 11.5 for HRV14, 8.4 for HRV21, and 12.1 for HRV71, respectively.

A Novel Series of Highly Potent Small Molecule Inhibitors of Rhinovirus Replication.

Human rhinoviruses (hRVs) are the main causative pathogen for common colds and are associated with the exacerbation of asthma. The wide variety in hRV serotypes has complicated the development of rhinovirus replication inhibitors. In the current investigation, we developed a novel series of benzothiophene derivatives and their analogues (6-8) that potently inhibit the replication of both hRV-A and hRV-B strains. Compound 6g inhibited the replication of hRV-B14, A21, and A71, with respective EC50 values of 0.083, 0.078, and 0.015 µM. The results of a time-of-addition study against hRV-B14 and hRV-A16 and resistant mutation analysis on hRV-B14 implied that 6g acts at the early stage of the viral replication process, interacting with viral capsid protein. A molecular docking study suggested that 6g has a capsid-binding mode similar to that of pleconaril. Finally, derivatives of 6 also displayed significant inhibition against poliovirus 3 (PV3) replication, implying their potential inhibitory activities against other enterovirus species.

CRISPR/Cas9-mediated gene knockout screens and target identification via whole-genome sequencing uncover host genes required for picornavirus infection.

Several groups have used genome-wide libraries of lentiviruses encoding small guide RNAs (sgRNAs) for genetic screens. In most cases, sgRNA expression cassettes are integrated into cells by using lentiviruses, and target genes are statistically estimated by the readout of sgRNA sequences after targeted sequencing. We present a new virus-free method for human gene knockout screens using a genome-wide library of CRISPR/Cas9 sgRNAs based on plasmids and target gene identification via whole-genome sequencing (WGS) confirmation of authentic mutations rather than statistical estimation through targeted amplicon sequencing. We used 30,840 pairs of individually synthesized oligonucleotides to construct the genome-scale sgRNA library, collectively targeting 10,280 human genes (i.e. three sgRNAs per gene). These plasmid libraries were co-transfected with a Cas9-expression plasmid into human cells, which were then treated with cytotoxic drugs or viruses. Only cells lacking key factors essential for cytotoxic drug metabolism or viral infection were able to survive. Genomic DNA isolated from cells that survived these challenges was subjected to WGS to directly identify CRISPR/Cas9-mediated causal mutations essential for cell survival. With this approach, we were able to identify known and novel genes essential for viral infection in human cells. We propose that genome-wide sgRNA screens based on plasmids coupled with WGS are powerful tools for forward genetics studies and drug target discovery.

Antiviral activity of micafungin against enterovirus 71.

BACKGROUND: Enterovirus 71 (EV71) is a major causative agent of hand-foot-mouth disease (HFMD) and also causes severe neurological complications, leading to fatality in young children. However, no effective therapy is currently available for the treatment of this infection. METHODS: We identified small-molecule inhibitors of EV71 from a screen of 968 Food and Drug Administration (FDA)-approved drugs, with which clinical application for EV71-associated diseases would be more feasible, using EV71 subgenomic replicon system. Primary hits were extensively evaluated for their antiviral activities in EV71-infected cells. RESULTS: We identified micafungin, an echinocandin antifungal drug, as a novel inhibitor of EV71. Micafungin potently inhibits the proliferation of EV71 as well as the replication of EV71 replicon in cells with a low micromolar IC50 (~5 µM). The strong antiviral effect of micafungin on EV71 replicon and the result from time-of-addition experiment demonstrated a targeting of micafungin on virion-independent intracellular process(es) during EV71 infection. Moreover, an extensive analysis excluded the involvement of 2C and 3A proteins, IRES-dependent translation, and also that of polyprotein processing in the antiviral effect of micafungin. CONCLUSIONS: Our research revealed a new indication of micafungin as an effective inhibitor of EV71, which is the first case reporting antiviral activity of micafungin, an antifungal drug.

Synergistic antiviral activity of gemcitabine and ribavirin against enteroviruses.

Enteroviruses are major causative agents of various human diseases, and some of them are currently considered to be an enormous threat to public health. However, no effective therapy is currently available for the treatment of these infections. We identified gemcitabine, a nucleoside-analog drug used for cancer treatment, from a screen of bioactive chemicals as a novel inhibitor of coxsackievirus B3 (CVB3) and enterovirus 71 (EV71). Gemcitabine potently inhibited the proliferation of CVB3 and EV71, as well as the replication of CVB3 and EV71 replicons, in cells with a low micromolar IC50 (1-5 µM). Its strong inhibitory effect was also observed in cells infected with human rhinoviruses, demonstrating broad-spectrum antiviral effects on enteroviruses. Mechanistically, an extensive analysis excluded the involvement of 2C, 3A, IRES-dependent translation, and also that of polyprotein processing in the antiviral effects of gemcitabine. Importantly, gemcitabine in combination with ribavirin, an antiviral drug currently being used against a few RNA viruses, exhibited a synergistic antiviral effect on the replication of CVB3 and EV71 replicons. Consequently, our results clearly demonstrate a new indication for gemcitabine as an effective broad-spectrum inhibitor of enteroviruses and strongly suggest a new therapeutic strategy using gemcitabine alone or in combination with ribavirin for the treatment of various diseases associated with enterovirus infection.

Synthesis and anti-influenza virus activity of 4-oxo- or thioxo-4,5-dihydrofuro[3,4-c]pyridin-3(1H)-ones.

A target-free approach was applied to discover anti-influenza viral compounds, where influenza infected Madin-Darby canine kidney cells were treated 7500 different small organic chemicals individually and reduction of virus-induced cytopathic effect was measured. One of the hit compounds was (Z)-1-((5-fluoro-1H-indol-3-yl)methylene)-6-methyl-4-thioxo-4,5-dihydrofuro[3,4-c]pyridin-3(1H)-one (15a) with half-maximal effective concentrations of 17.4-21.1µM against influenza A/H1N1, A/H3N2 and B viruses without any cellular toxicity at 900µM. To investigate the structure-activity relationships, two dozens of the hit analogs were synthesized. Among them, 15g, 15j, 15q, 15s, 15t and 15x had anti-influenza viral activity comparable or superior to that of the initial hit. The anti-influenza viral compounds efficiently suppressed not only viral protein level of the infected cells but also production of viral progeny in the culture supernatants in a dose-dependent manner. Based on a mode-of-action study, they did not affect virus entry or RNA replication. Instead, they suppressed viral neuraminidase activity. This study is the first to demonstrate that dihydrofuropyridinones could serve as lead compounds for the discovery of alternative influenza virus inhibitors.

Zoonotic encephalitides caused by arboviruses: transmission and epidemiology of alphaviruses and flaviviruses.

In this review, we mainly focus on zoonotic encephalitides caused by arthropod-borne viruses (arboviruses) of the families Flaviviridae (genus Flavivirus) and Togaviridae (genus Alphavirus) that are important in both humans and domestic animals. Specifically, we will focus on alphaviruses (Eastern equine encephalitis virus, Western equine encephalitis virus, Venezuelan equine encephalitis virus) and flaviviruses (Japanese encephalitis virus and West Nile virus). Most of these viruses were originally found in tropical regions such as Africa and South America or in some regions in Asia. However, they have dispersed widely and currently cause diseases around the world. Global warming, increasing urbanization and population size in tropical regions, faster transportation and rapid spread of arthropod vectors contribute in continuous spreading of arboviruses into new geographic areas causing reemerging or resurging diseases. Most of the reemerging arboviruses also have emerged as zoonotic disease agents and created major public health issues and disease epidemics.

Inhibition of influenza virus internalization by (-)-epigallocatechin-3-gallate.

(-)-Epigallocatechin-3-gallate (EGCG), one of the major flavonoid components of green tea, is known to have a broad antiviral activity against several enveloped viruses, including the influenza virus. However, its mode of action and the mechanism that allows it to target influenza virus molecules have not been fully elucidated. Thus, this study investigated the molecular mechanism by which EGCG suppresses influenza virus infections. EGCG was found to block an early step in the influenza viral life cycle, but it did not affect viral adsorption to target cells or viral RNA replication. However, EGCG inhibited hemifusion events between virus particles and the cellular membrane by reducing the viral membrane integrity, thereby resulting in the loss of the cell penetration capacity of the influenza virus. EGCG also marginally suppressed the viral and nonviral neuraminidase (NA) activity in an enzyme-based assay system. In conclusion, it is suggested that the anti-influenza viral efficacy of EGCG is attributable to damage to the physical properties of the viral envelope and partial inhibition of the NA surface glycoprotein. These results may facilitate future investigations of the antiviral activity of EGCG against other enveloped viruses as well as influenza virus.

Epimeric methylsulfinyladenosine derivatives from the marine ascidian Herdmania momus.

Investigation of the secondary metabolites of the ascidian Herdmania momus led to the isolation and characterization of four new nucleoside derivatives (1-4). Structural studies showed that these derivatives represent a series of rare methylsulfinyladenosine derivatives of interconvertible transesterification isomers and/or sulfinyl epimers. The antiviral activities of these rare nucleosides were evaluated against a series of human pathogenic viruses.

Efficient synthesis of 3H,3'H-spiro[benzofuran-2,1'-isobenzofuran]-3,3'-dione as novel skeletons specifically for influenza virus type B inhibition.

An efficient and novel two step synthetic procedure to prepare various substituted 3H,3'H-spiro[benzofuran-2,1'-isobenzofuran]-3,3'-diones A, was established from very simple and easily available starting materials. The developed method is a robust and general approach for the synthesis of these structures. The prepared compounds were tested against influenza virus type A viz., A/Taiwan/1/86 (H1N1), A/Hong Kong/8/68 (H3N2) and type B viz., B/Panama/45/90, B/Taiwan/2/62, B/Lee/40, B/Brisbane/60/2008. Among 31 compounds tested, some of them showed good activity (selective index values >10) against these influenza viruses preferentially for type B. The most active compound 3b showed activity in 3.0-16.1 µM range with a selectivity index value between 30 and 166 against these type B viruses, in which it was comparable to the antiviral agent favipiravir. Also, 3b is found to be inactive against other enveloped viruses (viz., HIV and HSV) showing its specificity for influenza viruses.

In vitro inhibition of influenza A virus infection by marine microalga-derived sulfated polysaccharide p-KG03.

The sulfated polysaccharide, p-KG03, purified from the marine microalga, Gyrodinium impudium, is a unique compound comprising homogenous galactose units conjugated to uronic acid and sulfated groups. Although previous studies showed that p-KG03 suppresses tumor cell growth and infection by encephalomyocarditis virus, its effect against enveloped virus infection and the biological mechanism of action have not been elucidated. In this report, the inhibitory activity of p-KG03 against influenza virus was examined and compared with that of other sulfated polysaccharides (fucoidan and pentosan polysulfate) and antiviral agents (oseltamivir phosphate, oseltamivir carboxylate, amantadine, and ribavirin). The results of a cytopathic effect reduction assay using MDCK cells demonstrated that p-KG03 exhibited the 50% effective concentration (EC(50)) values of 0.19-0.48 µg/ml against influenza type A virus infection (selectivity index >200) but not all influenza type B viruses. Mechanism studies showed that inhibition of influenza virus replication was maximized when p-KG03 was added during or within 6 h after viral infection, suggesting that mainly the viral adsorption and internalization steps are targeted by this compound. The results of influenza virus binding assay to p-KG03 and fluorescence microscopy indicate that the antiviral activity of p-KG03 is directly associated with its interaction with viral particles. The sulfated polysaccharide p-KG03 is a potent and specific influenza A viral entry inhibitor and may be a candidate for antiviral drug development.

Synthesis of enantiomerically pure D- and L-bicyclo[3.1.0]hexenyl carbanucleosides and their antiviral evaluation.

Based upon the fact that L-nucleosides have been generally known to be less cytotoxic than D-counterparts, L-bicyclo[3.1.0]hexenyl carbanucleoside derivatives with a fixed north conformation were designed and synthesized by employing a novel synthetic strategy starting from (R)-epichlorohydrin in order to search for new anti-HIV agents with high potency and less cytotoxicity. A tandem alkylation, γ-lactonization, a chemoselective reduction of ester in the presence of γ-lactone functional group, a RCM reaction, and a Mitsunobu coupling reaction were used as key reactions. D-Counterpart nucleosides were also prepared according to the same synthetic method. Among the synthesized carbanucleosides, D-thymine nucleoside, D-2 and L-thymine nucleoside, L-2 exhibited excellent anti-HIV-1 and -2 activities, in MT-4 cells, which were higher than those of ddI, an anti-AIDS drug. Whereas D-2 exhibited high cytotoxicity in MT-4 cell lines, L-2 did not show any discernible cytotoxicity in all cell lines tested, reflecting that L-2 may be a good candidate for an anti-AIDS drug. L-2 also showed weak anti-HSV-2 activity without cytotoxicity. However, none of the synthesized nucleosides exhibited antiviral activities against RNA viruses including coxsakie, influenza, corona and polio viruses, maybe due to their 2',3'-dideoxy structure. Potent antiviral effects of D-2 and L-2 indicate that nucleosides belonging to a class of D4Ns can be an excellent candidate for anti-DNA virus agents. This research strongly supports L-nucleosides of a class of D4Ns to be a very promising candidate for antiviral agents due to its low cytotoxicity and a good antiviral activity.

Design and synthesis of 3'-fluoropenciclovir analogues as antiviral agents.

Based on fluorine switch approach, a series of 3'-fluoropenciclovir analogues with different purine and pyrimidine bases were designed and synthesized. Direct reduction of beta-fluoroester to the corresponding 3-fluoroalcohol provided an easy and new entry pathway towards the synthesis of 3'-fluoropenciclovir analogues. The synthesized 3'-fluoropenciclovir analogues were evaluated for their antiviral activities against the poliovirus, HSV-1, HSV-2 and HIV.

N1-Heterocyclic pyrimidinediones as non-nucleoside inhibitors of HIV-1 reverse transcriptase.

A series of N1-heterocyclic pyrimidinediones were extensively evaluated as HIV-1 non-nucleoside reverse transcriptase inhibitors (NNRTIs). Inhibitor 1 is active against NNRTI-resistant viruses including RT mutant K103N. The co-crystal structure of inhibitor 1 with HIV-1 RT revealed that H-bonds are formed with K101 and K103. Efforts to improve the suboptimal pharmacokinetic profile of 1 resulted in the discovery of compound 13, which represents the lead compound in this series with improved pharmacokinetics and similar potency as inhibitor 1.

Caffeoylglycolic and caffeoylamino acid derivatives, halfmers of L-chicoric acid, as new HIV-1 integrase inhibitors.

Human immunodeficiency virus (HIV) integrase (IN) catalyzes the integration of HIV DNA copy into the host cell DNA. L-Chicoric acid (1) has been found to be one of the most potent HIV-1 integrase inhibitor. Caffeoylglycolic and caffeoylamino acid derivatives' halfmeric structures of L-chicoric acid 2 were synthesized for the purpose of simplifying the structure of L-chicoric acid. Among synthesized, compounds 2c and 3f showed HIV-1 IN inhibitory activities with IC(50) values of 10.5 and 12.0 microM, respectively, comparable to that of parent compound L-chicoric acid (IC(50)=15.7 microM).

Intracellular uptake of thymidine and antiherpetic drugs for thymidine kinase-deficient mutants of herpes simplex virus type 1.

The influence of the thymidine (Thd) kinase (TK) of herpes simplex virus type 1 (HSV-1) on the intracellular uptake and anabolism of nucleosides has been investigated. To compare the differences between the TK-positive (TK(+)) and TK-deficient strains, acyclovir (ACV)-resistant strains were cloned from a cell culture and classified into 2 groups, viz. the TK-partial (TK(p)) and TK-negative (TK(-)). The cellular uptake of thymidine was highly dependent on the viral TK (vTK) activity. The TK(+) strain showed the highest level of intracellular thymidine uptake, the TK(p) strain a moderate level, which varied from strain to strain, and the TK(-) and mock strains showed little uptake. The inhibition of viral replication by ACV, ganciclovir (GCV) and penciclovir (PCV) did not decrease the Thd uptake at all. On the contrary, a notable increase found to be induced by ACV. The influence of the vTK on the uptake of GCV or PCV was much greater than that of ACV. The metabolism was generally less dependent on the vTK activity than the influx. The influx and phosphorylation rates of GCV and PCV were dependent on the substrate specificity of the vTK.

Antiviral effects of sulfated exopolysaccharide from the marine microalga Gyrodinium impudicum strain KG03.

The sulfated exopolysaccharide p-KG03, which is produced by the marine microalga Gyrodinium impudicum strain KG03, exhibited impressive antiviral activity in vitro (EC50 = 26.9 micro g/ml) against the encephalomyocarditis virus (EMCV). Depending on the p-KG03 concentration, the development of cytopathic effects in EMCV-infected HeLa cells was either inhibited completely or slowed. Moreover, p-KG03 did not show any cytotoxic effects on HeLa cells, even at concentrations up to 1000 microg/ml. The polysaccharide was purified by repeated precipitation in ethanol, followed by gel filtration. The p-KG03 polysaccharide had a molecular weight of 1.87 x 10(7), and was characterized as a homopolysaccharide of galactose with uronic acid (2.96% wt/wt) and sulfate groups (10.32% wt/wt). The biological activities of p-KG03 suggest that sulfated metabolites from marine organisms are a rich source of antiviral agents. This is the first reported marine source of antiviral sulfated polysaccharides against EMCV. The p-KG03 polysaccharide may be useful in the development of marine bioactive exopolysaccharide for biotechnological and pharmaceutical products.

Synthesis of (Z)-(1-fluoro-2-hydroxymethylcyclopropylmethyl)purines.

(Z)-(1-fluoro-2-hydroxymethylcyclopropylmethyl)purines were designed, synthesized and evaluated their antiviral activity against poliovirus, HSV, and HIV.