Replication-Competent Influenza Virus and Respiratory Syncytial Virus Luciferase Reporter Strains Engineered for Co-Infections Identify Antiviral Compounds in Combination Screens

Yan, Dan; Weisshaar, Marco; Lamb, K.M.; Chung, Hokyung K.; Lin, Michael Z.; Plemper, Richard K.

doi:10.1021/acs.biochem.5b00623

Cited by 36 publications

(40 citation statements)

References 85 publications

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“…These findings were clearly documented by confocal micro- (44,45). In addition, WD-PBEC cultures were used to confirm that this process also occurs in a more representative model of primary airway epithelial cells.…”

Section: Discussionmentioning

confidence: 63%

Antibody-Induced Internalization of the Human Respiratory Syncytial Virus Fusion Protein

Leemans

Schryver

Gucht

et al. 2017

J Virol

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Respiratory syncytial virus (RSV) infections remain a major cause of respiratory disease and hospitalizations among infants. Infection recurs frequently and establishes a weak and short-lived immunity. To date, RSV immunoprophylaxis and vaccine research is mainly focused on the RSV fusion (F) protein, but a vaccine remains elusive. The RSV F protein is a highly conserved surface glycoprotein and is the main target of neutralizing antibodies induced by natural infection. Here, we analyzed an internalization process of antigen-antibody complexes after binding of RSV-specific antibodies to RSV antigens expressed on the surface of infected cells. The RSV F protein and attachment (G) protein were found to be internalized in both infected and transfected cells after the addition of either RSV-specific polyclonal antibodies (PAbs) or RSV glycoprotein-specific monoclonal antibodies (MAbs), as determined by indirect immunofluorescence staining and flow-cytometric analysis. Internalization experiments with different cell lines, well-differentiated primary bronchial epithelial cells (WD-PBECs), and RSV isolates suggest that antibody internalization can be considered a general feature of RSV. More specifically for RSV F, the mechanism of internalization was shown to be clathrin dependent. All RSV F-targeted MAbs tested, regardless of their epitopes, induced internalization of RSV F. No differences could be observed between the different MAbs, indicating that RSV F internalization was epitope independent. Since this process can be either antiviral, by affecting virus assembly and production, or beneficial for the virus, by limiting the efficacy of antibodies and effector mechanism, further research is required to determine the extent to which this occurs in vivo and how this might impact RSV replication.IMPORTANCE Current research into the development of new immunoprophylaxis and vaccines is mainly focused on the RSV F protein since, among others, RSV F-specific antibodies are able to protect infants from severe disease, if administered prophylactically. However, antibody responses established after natural RSV infections are poorly protective against reinfection, and high levels of antibodies do not always correlate with protection. Therefore, RSV might be capable of interfering, at least partially, with antibody-induced neutralization. In this study, a process through which surface-expressed RSV F proteins are internalized after interaction with RSVspecific antibodies is described. One the one hand, this antigen-antibody complex internalization could result in an antiviral effect, since it may interfere with virus par-

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Section: Discussionmentioning

confidence: 63%

Antibody-Induced Internalization of the Human Respiratory Syncytial Virus Fusion Protein

Leemans

Schryver

Gucht

et al. 2017

J Virol

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“…Based on coinfection of cells with independently traceable replication-competent IAV and respiratory syncytial virus (RSV) reporter strains expressing nano and firefly luciferases, respectively, this protocol is designed to simultaneously identify orthomyxovirus-specific, paramyxovirus-specific, and broad-spectrum likely host-directed hit candidates. Full assay validation against a small test set confirmed the suitability of this innovative approach for automated drug discovery (35).…”

mentioning

confidence: 76%

“…This first application of the myxovirus coinfection protocol (35) to a large open discovery library revealed the following distinct advantages of the strategy: robust assay parameters are maintained for each target strain under real screening conditions; the approach is highly resource and time effective; use of replication-competent reporter strains provides compatibility with a variety of host cell lines, including the human respiratory cells used in our study; and the simultaneous assessment of two distinct viral targets reliably eliminates undesirable cytotoxic compounds from the virus-specific hit pool. While the frequency of confirmed IAV inhibitors identified in this screen was relatively low, we have no indication to assume that this outcome may be linked to the coinfection protocol itself.…”

Section: Discussionmentioning

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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“…Guided by the past experience that pan-resistance-sensitive RSV entry inhibitors emerge readily in HTS campaigns, we have in recent work developed an innovative HTS design that counterselects against identifying additional chemical scaffolds sensitive to pan-resistance by including a signature resistance mutation into a recombinant RSV reporter strain 18 that also features a fusion protein derived from the line19 RSV isolate 19 . For automated detection of replication, this recombinant recRSV-A2-L19F D489E -fireSMASh strain expresses a firefly luciferase reporter from an additional transcription unit that is linked to an engineered small-molecule assisted shut off (SMASh) domain 20 , which significantly boosts the dynamic range of the assay, allowing effective assay miniaturization to 384-well plate format.…”

Section: Introductionmentioning

confidence: 99%

Identification of Non-Nucleoside Inhibitors of the Respiratory Syncytial Virus Polymerase Complex

et al. 2017

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Respiratory syncytial virus (RSV) represents a threat to infants, the elderly, and the immunocompromised. RSV entry blockers are in clinical trials, but escape mutations challenge their potential. In search of RSV inhibitors, we have integrated a signature resistance mutation into are combinant RSV virus and applied the strain to high-throughput screening. Counter screening of candidates returned 14 confirmed hits with activity in the nano- to low-micromolar range. All blocked RSV polymerase activity in minigenome assays. Compound 1a (GRP-74915) was selected for development based on activity (EC50=0.21 µM, selectivity index (SI) 40), and scaffold. Resynthesis confirmed potency of the compound, which suppressed viral RNA synthesis in infected cells. However, metabolic testing revealed a short half-life in the presence of mouse hepatocyte fractions. Metabolite tracking and chemical elaboration combined with 3D-quantitative structure-activity relationship modeling yielded analogs (i.e. 8n: EC50=0.06 µM, SI 500) that establish a platform for the development of a therapeutic candidate.

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Replication-Competent Influenza Virus and Respiratory Syncytial Virus Luciferase Reporter Strains Engineered for Co-Infections Identify Antiviral Compounds in Combination Screens

Cited by 36 publications

References 85 publications

Antibody-Induced Internalization of the Human Respiratory Syncytial Virus Fusion Protein

Antibody-Induced Internalization of the Human Respiratory Syncytial Virus Fusion Protein

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

Identification of Non-Nucleoside Inhibitors of the Respiratory Syncytial Virus Polymerase Complex

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