A Mechanistic Paradigm for Broad-Spectrum Antivirals that Target Virus-Cell Fusion

Vigant, Frédéric; Lee, Jihye; Hollmann, Axel; Tanner, Lukas B.; Ataman, Zeynep Akyol; Yun, Tatyana; Shui, Guanghou; Aguilar, Hector C.; Zhang, Dong; Meriwether, David; Roman-Sosa, Gleyder; Robinson, Lindsey R.; Juelich, Terry L.; Buczkowski, Hubert; Chou, Sunwen; Castanho, Miguel A. R. B.; Wolf, Mike C.; Smith, Jennifer K.; Banyard, Ashley C.; Kielian, Margaret; Reddy, Srinivasa T.; Wenk, Markus R.; Selke, Matthias; Santos, Nuno C.; Freiberg, Alexander N.; Jung, Michael E.; Lee, Benhur

doi:10.1371/journal.ppat.1003297

Cited by 94 publications

(154 citation statements)

References 57 publications

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“…Together, these data suggest that this compound has a broadspectrum mechanism of action that specifically disrupts viral membranes while sparing cellular membranes. During the course of our studies, a related compound was identified and characterized by others as a broad-spectrum compound that irreparably damages viral membranes (37,38), corroborating our data and validating the utility of our counterscreening approach for discovering broad-spectrum viral inhibitors. …”

Section: Incorporation Of M2 Ion Channels Into Vlpssupporting

confidence: 85%

Detection of Proton Movement Directly across Viral Membranes To Identify Novel Influenza Virus M2 Inhibitors

Sulli

Banik

Schilling

et al. 2013

J Virol

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The influenza virus M2 protein is a well-validated yet underexploited proton-selective ion channel essential for influenza virus infectivity. Because M2 is a toxic viral ion channel, existing M2 inhibitors have been discovered through live virus inhibition or medicinal chemistry rather than M2-targeted high-throughput screening (HTS), and direct measurement of its activity has been limited to live cells or reconstituted lipid bilayers. Here, we describe a cell-free ion channel assay in which M2 ion channels are incorporated into virus-like particles (VLPs) and proton conductance is measured directly across the viral lipid bilayer, detecting changes in membrane potential, ion permeability, and ion channel function. Using this approach in high-throughput screening of over 100,000 compounds, we identified 19 M2-specific inhibitors, including two novel chemical scaffolds that inhibit both M2 function and influenza virus infectivity. Counterscreening for nonspecific disruption of viral bilayer ion permeability also identified a broad-spectrum antiviral compound that acts by disrupting the integrity of the viral membrane. In addition to its application to M2 and potentially other ion channels, this technology enables direct measurement of the electrochemical and biophysical characteristics of viral membranes.T he M2 gene of influenza virus encodes a 97-amino-acid, homotetrameric, proton-selective ion channel necessary for influenza virus infectivity (1, 2). Activation of M2 is triggered by low pH in host cell endosomes, resulting in an influx of protons into the virus and acidification that releases the viral RNA into the host cell (2-4). M2 also helps to package viral RNA (5, 6) and regulate pH in the Golgi apparatus of infected cells during viral assembly (7). Two FDA-approved adamantane-based drugs, amantadine and rimantadine (2,8), are potent M2 inhibitors that can neutralize influenza virus in humans and other animals. However, the emergence of widespread highly virulent adamantane-resistant strains such as avian and swine influenza virus strains (9, 10) has prompted the need for newer antivirals. Drug discovery efforts targeting M2 have been difficult due to the toxicity of M2 in cells (11), the difficulty of reconstituting M2 in liposomes for largescale application (12, 13), and the labor-intensive, low-throughput electrophysiological approaches typically used to study ion channels.To address these challenges, we developed a rapid, cell-free assay to measure the movement of proton ions directly across the lipid bilayer of virus-like particles (VLPs). High-throughput screening (HTS) of M2-VLPs using Ͼ100,000 compounds identified 19 M2-specific inhibitors, including two novel chemical scaffolds, that inhibited both M2 function and influenza virus infectivity. Counterscreening for nonspecific disruption of carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) protonophore activity also identified a broad-spectrum antiviral compound that acts by disrupting the integrity of the viral membrane but that is not t...

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Section: Incorporation Of M2 Ion Channels Into Vlpssupporting

confidence: 85%

Detection of Proton Movement Directly across Viral Membranes To Identify Novel Influenza Virus M2 Inhibitors

Sulli

Banik

Schilling

et al. 2013

J Virol

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“…ecently, a few broad-spectrum antivirals have been described that target enveloped virus entry (1)(2)(3)(4)(5)(6)(7)(8)(9)(10). Antivirals that can target the biophysical process of virus-cell membrane fusion itself, rather than any particular viral protein, not only have the potential to be truly broad spectrum (against any enveloped virus) but also will likely have a high barrier to resistance (11)(12)(13)(14).…”

mentioning

confidence: 99%

“…Follow-up studies indicated that LJ001, our first generation of Broad-SAVE, and the oxazolidine-based JL series of compounds, our second generation Broad-SAVE, act as membrane-targeted photosensitizers: they generate singlet oxygen ( 1 O 2 ) in the plane of the membrane, and 1 O 2 -mediated lipid oxidation results in changes in the biophysical properties of the viral envelope that are not conducive to virus-cell membrane fusion (8). The salient point is that at antiviral concentrations, 1 O 2 -mediated lipid oxidation is not detrimental to metabolically active cell membranes.…”

mentioning

confidence: 99%

“…The salient point is that at antiviral concentrations, 1 O 2 -mediated lipid oxidation is not detrimental to metabolically active cell membranes. This is the key mechanistic paradigm that has never been explicitly stated and tested (15) until our recent studies (8,9).…”

mentioning

confidence: 99%

“…Indeed, dUY11 generated 1 O 2 in solution, as indicated by the oxidation of the 1 O 2 -specific trap 9,10-dimethylanthracene (DMA) (8) (Fig. 1B).…”

mentioning

confidence: 99%

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The Rigid Amphipathic Fusion Inhibitor dUY11 Acts through Photosensitization of Viruses

Vigant

Hollmann

Lee

et al. 2014

J Virol

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cRigid amphipathic fusion inhibitors (RAFIs) are lipophilic inverted-cone-shaped molecules thought to antagonize the membrane curvature transitions that occur during virus-cell fusion and are broad-spectrum antivirals against enveloped viruses (Broad-SAVE). Here, we show that RAFIs act like membrane-binding photosensitizers: their antiviral effect is dependent on light and the generation of singlet oxygen ( 1 O 2 ), similar to the mechanistic paradigm established for LJ001, a chemically unrelated class of Broad-SAVE. Photosensitization of viral membranes is a common mechanism that underlies these Broad-SAVE.

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Translation of Mycobacterium Survival Strategy to Develop a Lipo‐peptide based Fusion Inhibitor**

et al. 2021

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The entry of enveloped virus requires the fusion of viral and host cell membranes. An effective fusion inhibitor aiming at impeding such membrane fusion may emerge as a broad‐spectrum antiviral agent against a wide range of viral infections. Mycobacterium survives inside the phagosome by inhibiting phagosome–lysosome fusion with the help of a coat protein coronin 1. Structural analysis of coronin 1 and other WD40‐repeat protein suggest that the trp‐asp (WD) sequence is placed at distorted β‐meander motif (more exposed) in coronin 1. The unique structural feature of coronin 1 was explored to identify a simple lipo‐peptide sequence (myr‐WD), which effectively inhibits membrane fusion by modulating the interfacial order, water penetration, and surface potential. The mycobacterium inspired lipo‐dipeptide was successfully tested to combat type 1 influenza virus (H1N1) and murine coronavirus infections as a potential broad‐spectrum antiviral agent.

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A Mechanistic Paradigm for Broad-Spectrum Antivirals that Target Virus-Cell Fusion

Cited by 94 publications

References 57 publications

Detection of Proton Movement Directly across Viral Membranes To Identify Novel Influenza Virus M2 Inhibitors

Detection of Proton Movement Directly across Viral Membranes To Identify Novel Influenza Virus M2 Inhibitors

The Rigid Amphipathic Fusion Inhibitor dUY11 Acts through Photosensitization of Viruses

Translation of Mycobacterium Survival Strategy to Develop a Lipo‐peptide based Fusion Inhibitor**

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