qHTS Assay for Inhibitors of Influenza NS1 Protein Function: RT-PCR
Influenza is a world-wide public health problem and emerging forms of the virus have the potential to cause a pandemic of equal or greater magnitude to the outbreaks recorded in 1918, 1957 or 1968. Vaccine development is proceeding and there also exist two classes of anti-influenza compounds. However these therapeutic modalities are neither fully effective nor widely enough available to fulfill more ..
BioActive Compounds: 4
Influenza is a world-wide public health problem and emerging forms of the virus have the potential to cause a pandemic of equal or greater magnitude to the outbreaks recorded in 1918, 1957 or 1968. Vaccine development is proceeding and there also exist two classes of anti-influenza compounds. However these therapeutic modalities are neither fully effective nor widely enough available to fulfill global needs. In addition their potential usefulness against newly emergent strains is not known. Efforts are needed to develop novel agents against influenza virus, including broad-spectrum agents. Identification of small molecules that inhibit NS1 function either directly or by interfering with specific cellular pathways may be a key to increasing our defense against the virus.
We have miniaturized and optimized a cell based assay in which NS1 from influenza A is expressed in the budding yeast S. cerevisiae. NS1 is a multi-functional protein that counters the host innate immune response and facilitates viral versus cellular gene expression. Expression of NS1 causes a pronounced slow growth phenotype in yeast due to its intrinsic molecular activities. Small molecules that suppress the slow growth phenotype can be identified by a straightforward growth recovery assay using optical density (OD) as the measurement. The same yeast strain not expressing NS1 was used as positive control in the yeast growth recovery assay.
Hits resulting from the primary screen and other follow-up screens, were tested at 20 uM in this RT-PCR assay to measure induction of interferon-beta mRNA>
NIH Chemical Genomics Center [NCGC]
NIH Molecular Libraries Probe Centers Network [MLPCN]
MLPCN Grant: MH084878
Assay Submitter (PI): Daniel Engel, University of Virgina
MDCK cells were infected for 6 h with A/PR/8 or delNS1 at an MOI of 0.1 in the presence or absence of drug at 20 uM, and the total RNA was isolated by using RNeasy (Qiagen). For first-strand cDNA synthesis, 2 mg of total RNA was primed with random nanomers (New England Biolabs) at a final concentration of 2 muM. Reverse transcription was performed with 10 U of Moloney murine leukemia virus reverse transcriptase (New England Biolabs) mul-1 in the presence of 1 U of RNase inhibitor (New England Biolabs) mul-1. Thereafter, 1/20 volume of cDNA was used as a template for PCR (30 cycles). The following primer pairs were used: canine IFN-beta (accession no. NM_001135787), 5'-CCAGTTCCAGAAGGAGGACA-3' and 5'CCTGTTGTCCCAGGTGAA GT-3'; and canine beta-actin (GenBank accession no. XM_536230), 5'GGCATCCTGACCCT GAAGTA-3' and 5'GGGGTGTTGAAAGTCTCGAA-3'.
Compounds were considered "active" if a band was observed; compounds were considered "inactive" if no band was obsereved.
"Active compounds are assigned a score of 90, "inconclusive" 50, and "inactive" 10.
Data Table (Concise)