Meehyein Kim

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.

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.

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.

Identification and evaluation of potent Middle East respiratory syndrome coronavirus (MERS-CoV) 3CLPro inhibitors

ABSTRACT Middle East respiratory syndrome coronavirus (MERS‐CoV) causes severe acute respiratory illness with fever, cough and shortness of breath. Up to date, it has resulted in 1826 human infections, including 649 deaths. Analogous to picornavirus 3C protease (3Cpro), 3C‐like protease (3CLpro) is critical for initiation of the MERS‐CoV replication cycle and is thus regarded as a validated drug target. As presented here, our peptidomimetic inhibitors of enterovirus 3Cpro (6b, 6c and 6d) inhibited 3CLpro of MERS‐CoV and severe acute respiratory syndrome coronavirus (SARS‐CoV) with IC50 values ranging from 1.7 to 4.7 &mgr;M and from 0.2 to 0.7 &mgr;M, respectively. In MERS‐CoV‐infected cells, the inhibitors showed antiviral activity with EC50 values ranging from 0.6 to 1.4 &mgr;M, by downregulating the viral protein production in cells as well as reducing secretion of infectious viral particles into culture supernatants. They also suppressed other &agr;‐ and &bgr;‐CoVs from human and feline origin. These compounds exhibited good selectivity index (over 70 against MERS‐CoV) and could lead to the development of broad‐spectrum antiviral drugs against emerging CoVs and picornaviruses. Graphical abstract Figure. No Caption available. HighlightsAldehyde‐containing peptidomimetics were identified to be potent inhibitors against MERS‐CoV 3CLpro.The active inhibitor showed sub‐&mgr;M EC50 in killing MERS‐CoV.Compounds were also effective against other &agr; and &bgr;‐CoVs of both human and feline origin.We identified broad‐spectrum antiviral agents effective against both coronaviruses and picornaviruses.

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

Abstract 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.