Journal
BIOCHEMISTRY
Volume 54, Issue 36, Pages 5589-5604Publisher
AMER CHEMICAL SOC
DOI: 10.1021/acs.biochem.5b00623
Keywords
-
Categories
Funding
- Public Health Service from National Institute of Allergy and Infectious Diseases [AI071002, AI119196, HD079327]
- Eunice Kennedy Shriver National Institute of Child Health and Human Development
Ask authors/readers for more resources
Myxoviruses such as influenza A virus (IAV) and respiratory syncytial virus (RSV) are major human pathogens, mandating the development of novel therapeutics. To establish a high-throughput screening protocol for the simultaneous identification of pathogen- and host-targeted hit candidates against either pathogen or both, we have attempted coinfection of cells with IAV and RSV. However, viral replication kinetics were incompatible, RSV signal window was low, and an IAV-driven minireplicon reporter assay used in initial screens narrowed the host cell range and restricted the assay to single-cycle infections. To overcome these limitations, we developed an RSV strain carrying firefly luciferase fused to an innovative universal small-molecule assisted shut-off domain, which boosted assay signal window, and a hyperactive fusion protein that synchronized IAV and RSV reporter expression kinetics and suppressed the identification of RSV entry inhibitors sensitive to a recently reported RSV pan-resistance mechanism. Combined with a replication-competent recombinant IAV strain harboring nanoluciferase, the assay performed well on a human respiratory cell line and supports multicycle infections. Miniaturized to 384-well format, the protocol was validated through screening of a set of the National Institutes of Health Clinical Collection (NCC) in quadruplicate. These test screens demonstrated favorable assay parameters and reproducibility. Application to a LOPAC library of bioactive compounds in a proof-of-concept campaign detected licensed antimyxovirus therapeutics, ribavirin and the neuraminidase inhibitor zanamivir, and identified two unexpected RSV-specific hit candidates, Fenretinide and the opioid receptor antagonist BNTX-7. Hits were evaluated in direct and orthogonal dos- response counterscreens using a standard recRSV reporter strain expressing Renilla luciferase.
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available