New Small Molecule Entry Inhibitors Targeting Hemagglutinin-Mediated Influenza A Virus Fusion

Basu, Arnab; Antanasijevic, Aleksandar; Li, Bing; Mills, Debra M.; Ames, Jessica A.; Nash, Peter J.; Williams, John D.; Peet, Norton P.; Moir, Donald T.; Prichard, Mark N.; Keith, Kathy A.; Barnard, Dale L.; Caffrey, Michael; Rong, Lijun; Bowlin, Terry L.

doi:10.1128/jvi.01225-13

Cited by 75 publications

(122 citation statements)

References 63 publications

(87 reference statements)

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“…Resistance mutations located in the HA2 B-loop domain were previously reported for at least six structurally unrelated classes of small-molecule IAV entry inhibitors (45)(46)(47). Remarkably, our adaptations reveal alternative, cooperative escape strategies mediated by substitutions located in distinct domains of the protein.…”

Section: Viral Adaptation For Resistance Profilingmentioning

confidence: 72%

“…Notably, IAV entry inhibitors have emerged as the predominant inhibitor class in these campaigns; five compounds were reported previously, in addition to the three scaffolds identified in our study. Of the five compounds discovered previously, two (MBX 2546 and FA-617 [46,47]) are direct analogs of each other, and another (45) is chemically related, resulting in a total of six truly independent chemical scaffolds. Despite their chemical diversity, the antiviral profiles of these six compound classes are remarkably similar: the indication spectrum of each is limited to group 1 IAVs, all are sensitive to resistance mutations at B-loop position 58 or 59, and all are predicted to populate the same pocket near the membrane-distal end of the prefusion HA stalk.…”

Section: Discussionmentioning

confidence: 99%

“…Recently, in three screening campaigns, six different compounds were identified that all block entry of group 1 IAVs (45-47), supposedly through interaction with the same target pocket as that suggested by our docking exercises. One of these studies employed a lentivirus vector system pseudotyped with IAV HA, limiting the hit pool to inhibitors of IAV entry (47), but the other two campaigns were open by design, assessing either IAV-induced cytopathicity as a readout (46) or using a second-generation IAV reporter system (45). Considering the two open screens only combined with our study employing a third-generation reporter strain, over 1,100,000 drug-like compounds have been used to interrogate potential druggable targets of IAVs.…”

Section: Discussionmentioning

confidence: 99%

“…Due to the pocket location between two HA monomers, compounds populating this microdomain may prevent membrane fusion by stabilizing the HA prefusion conformation. All reported group 1 IAV entry inhibitors are proposed to bind in or near this microdomain (45)(46)(47), but predicted docking poses and the nature of key points of contact are unique for each scaffold, as illustrated by two-dimensional (2D) transformations that we generated for the docking poses of the three GRP hit scaffolds (Fig. 8D).…”

Section: Viral Adaptation For Resistance Profilingmentioning

confidence: 99%

“…The different trigger pH ranges of HA proteins harboring cooperative and single escape mutations suggest that there are different underlying mechanisms of resistance. With the aim of extracting a candidate docking pose of the compounds, we expressed and purified two soluble HAs with signature mutations of the different escape classes: to facilitate comparison with previous studies (45)(46)(47), the HA2 M59I substitution was chosen to represent the cluster of single escape mutations located in proximity to the membrane-distal stalk section, and the HA2 D85N/HA2 T154S combination was selected as a signature pair for cooperative resistance.…”

Section: Viral Adaptation For Resistance Profilingmentioning

confidence: 99%

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Identification and Characterization of Influenza Virus Entry Inhibitors through Dual Myxovirus High-Throughput Screening

Weisshaar

Cox

Morehouse

et al. 2016

J Virol

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Influenza A virus (IAV) infections cause major morbidity and mortality, generating an urgent need for novel antiviral therapeutics. We recently established a dual myxovirus high-throughput screening protocol that combines a fully replication-competent IAV-WSN strain and a respiratory syncytial virus reporter strain for the simultaneous identification of IAV-specific, paramyxovirus-specific, and broad-spectrum inhibitors. In the present study, this protocol was applied to a screening campaign to assess a diverse chemical library with over 142,000 entries. Focusing on IAV-specific hits, we obtained a hit rate of 0.03% after cytotoxicity testing and counterscreening. Three chemically distinct hit classes with nanomolar potency and favorable cytotoxicity profiles were selected. Time-of-addition, minigenome, and viral entry studies demonstrated that these classes block hemagglutinin (HA)-mediated membrane fusion. Antiviral activity extends to an isolate from the 2009 pandemic and, in one case, another group 1 subtype. Target identification through biolayer interferometry confirmed binding of all hit compounds to HA. Resistance profiling revealed two distinct escape mechanisms: primary resistance, associated with reduced compound binding, and secondary resistance, associated with unaltered binding. Secondary resistance was mediated, unusually, through two different pairs of cooperative mutations, each combining a mutation eliminating the membrane-proximal stalk N-glycan with a membrane-distal change in HA1 or HA2. Chemical synthesis of an analog library combined with in silico docking extracted a docking pose for the hit classes. Chemical interrogation spotlights IAV HA as a major druggable target for small-molecule inhibition. Our study identifies novel chemical scaffolds with high developmental potential, outlines diverse routes of IAV escape from entry inhibition, and establishes a path toward structure-aided lead development. IMPORTANCEThis study is one of the first to apply a fully replication-competent third-generation IAV reporter strain to a large-scale highthroughput screen (HTS) drug discovery campaign, allowing multicycle infection and screening in physiologically relevant human respiratory cells. A large number of potential druggable targets was thus chemically interrogated, but mechanistic characterization, positive target identification, and resistance profiling demonstrated that three chemically promising and structurally distinct hit classes selected for further analysis all block HA-mediated membrane fusion. Viral escape from inhibition could be achieved through primary and secondary resistance mechanisms. In silico docking predicted compound binding to a microdomain located at the membrane-distal site of the prefusion HA stalk that was also previously suggested as a target site for chemically unrelated HA inhibitors. This study identifies an unexpected chemodominance of the HA stalk microdomain for smallmolecule inhibitors in IAV inhibitor screening campaigns and highlights a novel mechanism ...

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Section: Viral Adaptation For Resistance Profilingmentioning

confidence: 72%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Viral Adaptation For Resistance Profilingmentioning

confidence: 99%

Section: Viral Adaptation For Resistance Profilingmentioning

confidence: 99%

See 3 more Smart Citations

Identification and Characterization of Influenza Virus Entry Inhibitors through Dual Myxovirus High-Throughput Screening

Weisshaar

Cox

Morehouse

et al. 2016

J Virol

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Retroviral Pseudotypes – From Scientific Tools to Clinical Utility

Temperton

Wright

Scott

2015

Encyclopedia of Life Sciences

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Retroviral pseudotypes are important research and diagnostic tools for basic and clinical virology studies, facilitating the detailed investigation of individual genes, cellular receptors, antibody responses, serosurveillance and antiviral therapies. Importantly, pseudotypes enable the study of highly pathogenic viruses, without the need for high containment. Their use as gene therapy vectors is widely documented, but other uses, once less well known, are becoming more prominent. The substitution of envelope proteins expressed on the virion surface enables pseudotypes to be employed as surrogates for wildtype viruses in antibody neutralisation or antiviral screening assays and for the study of cell–virus receptor interactions. In addition, they are increasingly being utilised as vaccine immunogens, expressing the antigen either on the particle surface or as a transfer gene for cellular expression. These studies demonstrate the potential for using pseudotypes for both scientific and clinical applications. Key Concepts Students will learn: What are retroviral pseudotypes. How they are constructed and titrated. The common elements that comprise a retroviral pseudotype. How retroviral pseudotypes can be used to deliver genes. How they can be used as tools for the study of viral attachment, entry and receptor identification. About using pseudotypes as surrogate viruses in antibody neutralisation assays. The use of pseudotypes to measure vaccine immunogenicity and screen for antiviral activity and resistance. How pseudotypes can be used as experimental vaccines.

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Non‐Toxic Virucidal Macromolecules Show High Efficacy Against Influenza Virus Ex Vivo and In Vivo

et al. 2020

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Influenza is one of the most widespread viral infections worldwide and represents a major public health problem. The risk that one of the next pandemics is caused by an influenza strain is high. It is important to develop broad‐spectrum influenza antivirals to be ready for any possible vaccine shortcomings. Anti‐influenza drugs are available but they are far from ideal. Arguably, an ideal antiviral should target conserved viral domains and be virucidal, that is, irreversibly inhibit viral infectivity. Here, a new class of broad‐spectrum anti‐influenza macromolecules is described that meets these criteria and display exceedingly low toxicity. These compounds are based on a cyclodextrin core modified on its primary face with long hydrophobic linkers terminated either in 6'sialyl‐N‐acetyllactosamine (6’SLN) or in 3’SLN. SLN enables nanomolar inhibition of the viruses while the hydrophobic linkers confer irreversibility to the inhibition. The combination of these two properties allows for efficacy in vitro against several human or avian influenza strains, as well as against a 2009 pandemic influenza strain ex vivo. Importantly, it is shown that, in mice, one of the compounds provides therapeutic efficacy when administered 24 h post‐infection allowing 90% survival as opposed to no survival for the placebo and oseltamivir.

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New Small Molecule Entry Inhibitors Targeting Hemagglutinin-Mediated Influenza A Virus Fusion

Cited by 75 publications

References 63 publications

Identification and Characterization of Influenza Virus Entry Inhibitors through Dual Myxovirus High-Throughput Screening

Identification and Characterization of Influenza Virus Entry Inhibitors through Dual Myxovirus High-Throughput Screening

Retroviral Pseudotypes – From Scientific Tools to Clinical Utility

Non‐Toxic Virucidal Macromolecules Show High Efficacy Against Influenza Virus Ex Vivo and In Vivo

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