Dual Myxovirus Screen Identifies a Small-Molecule Agonist of the Host Antiviral Response

Yan, Dan; Krumm, Stefanie A.; Sun, Aiming; Steinhauer, David A.; Luo, Ming; Moore, Martin L.; Plemper, Richard K.

doi:10.1128/jvi.01425-13

Cited by 18 publications

(38 citation statements)

References 70 publications

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“…To identify novel anti-RSV drug candidates, we screened a 10,000-entry small-molecule library against a previously generated recombinant (rec) RSV strain harboring an additional transcription unit encoding for renilla luciferase (32,33). Applying recently established assay conditions for automated antiparamyxovirus drug screens (33), this exercise returned a hit candidate pool of 17 compounds, each with a primary screening score exceeding 10 times the overall SD of the assay.…”

Section: Resultsmentioning

confidence: 99%

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Cross-resistance mechanism of respiratory syncytial virus against structurally diverse entry inhibitors

Yan

Lee

Thakkar

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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109

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Respiratory syncytial virus (RSV) is a leading pediatric pathogen that is responsible for a majority of infant hospitalizations due to viral disease. Despite its clinical importance, no vaccine prophylaxis against RSV disease or effective antiviral therapeutic is available. In this study, we established a robust high-throughput drug screening protocol by using a recombinant RSV reporter virus to expand the pool of RSV inhibitor candidates. Mechanistic characterization revealed that a potent newly identified inhibitor class blocks viral entry through specific targeting of the RSV fusion (F) protein. Resistance against this class was induced and revealed overlapping hotspots with diverse, previously identified RSV entry blockers at different stages of preclinical and clinical development. A structural and biochemical assessment of the mechanism of unique, broad RSV cross-resistance against structurally distinct entry inhibitors demonstrated that individual escape hotspots are located in immediate physical proximity in the metastable conformation of RSV F and that the resistance mutations lower the barrier for prefusion F triggering, resulting in an accelerated RSV entry kinetics. One resistant RSV recombinant remained fully pathogenic in a mouse model of RSV infection. By identifying molecular determinants governing the RSV entry machinery, this study spotlights a molecular mechanism of broad RSV resistance against entry inhibition that may affect the impact of diverse viral entry inhibitors presently considered for clinical use and outlines a proactive design for future RSV drug discovery campaigns.antiviral drugs | drug resistance

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Section: Resultsmentioning

confidence: 99%

“…Applying recently established assay conditions for automated antiparamyxovirus drug screens (33), this exercise returned a hit candidate pool of 17 compounds, each with a primary screening score exceeding 10 times the overall SD of the assay. (Fig.…”

Section: Resultsmentioning

confidence: 99%

Cross-resistance mechanism of respiratory syncytial virus against structurally diverse entry inhibitors

Yan

Lee

Thakkar

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

109

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“…Serial dilutions of cherry-picked hit candidates were tested against a recVSV-nanoLuc strain (MOI ϭ 0.02) generated for the counterscreen and a previously developed recRSV-A2-renilla strain (31,38). Reporter signals were determined after 24 h (recVSVnanoLuc) or 48 h (recRSV-A2-renilla) of incubation at 37°C.…”

Section: Methodsmentioning

confidence: 99%

“…More recently, several generations of reporter systems were developed to assess virus replication, and these provide a quantitative readout and support miniaturization below the level of the 96-wellplate format (29). First-generation reporter assays employed a plasmid-based minigenome luciferase reporter driven by superinfection of transfected cells with influenza virus (30,31). However, the suitability of this approach for HTS was limited by the laborintensive transfection of target cells, a narrow range of suitable target cell lines, and a restriction to single-cycle infection at a high multiplicity of infection (MOI) for robust reporter expression, which prevents the discovery of late-acting compounds.…”

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confidence: 99%

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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“…Efficient heterotrimer formation and the complete lack of F trypsin bioactivity in the absence of exogenous trypsin allowed the functional assessment of F trimers harboring a single HR-B domain disulfide bond through the generation of transcomplementation fusion profiles. We used a transient kinetic cell-to-cell fusion assay (39,40) that is based on dualsplit eGFP-luciferase chimeras to determine maximal fusion rates for a panel of different F trypsin :F cysteine ratios for each of the F cysteine mutants (Fig. S7).…”

Section: Effect Of the Engineered Bonds On The Conformational Stabilimentioning

confidence: 99%

Efficient replication of a paramyxovirus independent of full zippering of the fusion protein six-helix bundle domain

Brindley¹,

Plattet²,

Plemper³

2014

Proc. Natl. Acad. Sci. U.S.A.

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Enveloped viruses such as HIV and members of the paramyxovirus family use metastable, proteinaceous fusion machineries to merge the viral envelope with cellular membranes for infection. A hallmark of the fusogenic glycoproteins of these pathogens is refolding into a thermodynamically highly stable fusion core structure composed of six antiparallel α-helices, and this structure is considered instrumental for pore opening and/or enlargement. Using a paramyxovirus fusion (F) protein, we tested this paradigm by engineering covalently restricted F proteins that are predicted to be unable to close the six-helix bundle core structure fully. Several candidate bonds formed efficiently, resulting in F trimers and higher-order complexes containing covalently linked dimers. The engineered F complexes were incorporated into recombinant virions efficiently and were capable of refolding into a postfusion conformation without temporary or permanent disruption of the disulfide bonds. They efficiently formed fusion pores based on virus replication and quantitative cell-to-cell and virus-to-cell fusion assays. Complementation of these F mutants with a monomeric, fusion-inactive F variant enriched the F oligomers for heterotrimers containing a single disulfide bond, without affecting fusion complementation profiles compared with standard F protein. Our demonstration that complete closure of the fusion core does not drive paramyxovirus entry may aid the design of strategies for inhibiting virus entry. membrane fusion | measles virus M embrane fusion is an essential process in eukaryotic cell biology. Examples range from fusion of lipid vesicles to sustain intracellular transport along secretory and endocytotic pathways to cell fusion in fertilization and development and to the fusion of viral with cellular membranes resulting in viral infection. Because there are several high-energy barriers (1), lipid membranes do not merge spontaneously under physiological conditions and therefore require the aid of auxiliary proteins to induce membrane stress and lower the activation energy for lipid merger. For vesicular transport, for instance, soluble N-ethylmaleimide-sensitive factor-activating protein receptor (SNARE) proteins resident in both the target and donor membrane mediate the process (2). Two fundamental differences distinguish cellular fusion machineries from viral fusogenic membrane glycoproteins (vFMGs), which mediate the entry of enveloped viruses by the fusion of the envelope with cellular membranes: (i) vFMGs do not require adapter proteins but insert a fusion peptide domain into the target membrane; and (ii) vFMGs fold into metastable prefusion conformations, obviating the dependence on external energy sources. Three distinct types of viral fusion (F) proteins have been classified: class I vFMGs are found, among others, on influenza virus, retroviruses, and paramyxoviruses; class II proteins are present on alpha-and flaviviruses; and class III proteins mediate herpes-and rhabdovirus entry (reviewed in ref.3).Once the fusion p...

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Dual Myxovirus Screen Identifies a Small-Molecule Agonist of the Host Antiviral Response

Cited by 18 publications

References 70 publications

Cross-resistance mechanism of respiratory syncytial virus against structurally diverse entry inhibitors

Cross-resistance mechanism of respiratory syncytial virus against structurally diverse entry inhibitors

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

Efficient replication of a paramyxovirus independent of full zippering of the fusion protein six-helix bundle domain

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