Discovery of 4′-Chloromethyl-2′-deoxy-3′,5′-di-<i>O</i>-isobutyryl-2′-fluorocytidine (ALS-8176), A First-in-Class RSV Polymerase Inhibitor for Treatment of Human Respiratory Syncytial Virus Infection

Wang, Guangyi; Deval, Jérôme; Ji, Hong; Dyatkina, Natalia; Prhavc, Marija; Taylor, Joshua; Fung, Amy; Zhang, Jin; Stevens, Sarah; Serebryany, Vladimir; Liu, Jyanwei; Zhang, Qingling; Tam, Yun K.; Chanda, Sushmita; Smith, David B.; Symons, Julian; Blatt, Lawrence M.; Beigelman, Leo

doi:10.1021/jm5017279

Cited by 115 publications

(131 citation statements)

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“…The 50% and 90% effective concentrations (EC 50 and EC 90 ) were assessed by four-parameter variable-slope regression modeling. The statistical significance of differences between two sample groups was assessed by two-way analysis of variance (ANOVA; Prism 6) in combination with Sidak's multiple-comparison posttest as specified in the figure legends.…”

Section: Discussionmentioning

confidence: 99%

“…Cells were infected with IAV-WSNnanoLuc (MOI ϭ 0.02) in the presence of compound at 10ϫ EC 50 . Viral growth was determined based on luciferase activity, and compound concentrations were increased to a maximum of 20 M when robust replication was detected.…”

Section: Discussionmentioning

confidence: 99%

“…Small-molecule viral entry inhibitors have proven efficacious against different viral targets in clinical and preclinical settings (50)(51)(52). In the past few decades, a number of influenza virus fusion blockers were described that originated from structure- aided drug discovery or screening campaigns (53)(54)(55)(56)(57)(58).…”

Section: Discussionmentioning

confidence: 99%

“…Raw data for all dose-response screens were analyzed according to the following formula: % inhibition ϭ (X Sample Ϫ X Min )/(X Max Ϫ X Min ) ϫ 100, with X Min representing the average for the positive-control wells and X Max the average for the negativecontrol wells. Four-parameter variable-slope regression was applied to determine 50% effective (EC 50 ) and cytotoxic (CC 50 ) concentrations.…”

Section: Methodsmentioning

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: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

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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“…Thus far, there are no licensed vaccines, although there are multiple vaccine candidates undergoing clinical trials (3). Development of antivirals against RSV is also an active field of research and clinical development (4)(5)(6).…”

mentioning

confidence: 99%

Respiratory Syncytial Virus Attachment Glycoprotein Contribution to Infection Depends on the Specific Fusion Protein

Meng

Hotard

Currier

et al. 2016

J Virol

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Human respiratory syncytial virus (RSV) is an important pathogen causing acute lower respiratory tract disease in children. The RSV attachment glycoprotein (G) is not required for infection, as G-null RSV replicates efficiently in several cell lines. Our laboratory previously reported that the viral fusion (F) protein is a determinant of strain-dependent pathogenesis. Here, we hypothesized that virus dependence on G is determined by the strain specificity of F. We generated recombinant viruses expressing G and F, or null for G, from the laboratory A2 strain (Katushka RSV-A2GA2F [kRSV-A2GA2F] and kRSV-GstopA2F) or the clinical isolate A2001/2-20 (kRSV-2-20G2-20F and kRSV-Gstop2-20F). We quantified the virus cell binding, entry kinetics, infectivity, and growth kinetics of these four recombinant viruses in vitro. RSV expressing the 2-20 G protein exhibited the greatest binding activity. Compared to the parental viruses expressing G and F, removal of 2-20 G had more deleterious effects on binding, entry, infectivity, and growth than removal of A2 G. Overall, RSV expressing 2-20 F had a high dependence on G for binding, entry, and infection. IMPORTANCERSV is the leading cause of childhood acute respiratory disease requiring hospitalization. As with other paramyxoviruses, two major RSV surface viral glycoproteins, the G attachment protein and the F fusion protein, mediate virus binding and subsequent membrane fusion, respectively. Previous work on the RSV A2 prototypical strain demonstrated that the G protein is functionally dispensable for in vitro replication. This is in contrast to other paramyxoviruses that require attachment protein function as a prerequisite for fusion. We reevaluated this requirement for RSV using G and F proteins from clinical isolate 2-20. Compared to the laboratory A2 strain, the G protein from 2-20 had greater contributions to virus binding, entry, infectivity, and in vitro growth kinetics. Thus, the clinical isolate 2-20 F protein function depended more on its G protein, suggesting that RSV has a higher dependence on G than previously thought. H uman respiratory syncytial virus (hRSV or RSV) causes an annual global 3.4 million estimated severe acute lower respiratory tract infections (ALRI) in children younger than 5 years of age (1). In the United States, about 132,000 to 172,000 children younger than 5 years of age are hospitalized due to RSV every year (2). Thus far, there are no licensed vaccines, although there are multiple vaccine candidates undergoing clinical trials (3). Development of antivirals against RSV is also an active field of research and clinical development (4-6).RSV is a member of the Paramyxoviridae family, Pneumovirus genus. Members of the paramyxovirus family encode two major glycoproteins important early during infection for attachment to the host cell and the subsequent entry process. Paramyxovirus fusion mediated by the viral fusion (F) protein is generally initiated by interaction with the homologous attachment protein upon receptor engagement (reviewed ...

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3-Nitro-1,2,4-triazole

Kumar

Lee

2017

Encyclopedia of Reagents for Organic Synthesis

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Discovery of 4′-Chloromethyl-2′-deoxy-3′,5′-di-O-isobutyryl-2′-fluorocytidine (ALS-8176), A First-in-Class RSV Polymerase Inhibitor for Treatment of Human Respiratory Syncytial Virus Infection

Cited by 115 publications

References 38 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

Respiratory Syncytial Virus Attachment Glycoprotein Contribution to Infection Depends on the Specific Fusion Protein

3-Nitro-1,2,4-triazole

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