qHTS Assay for Inhibitors of Influenza NS1 Protein Function: SAR Using NS1 and pYES strains
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: 5
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 using the same yeast strain that was used in the primary assay (NS1) but as control, were compared to a strain that lacks NS1 (pYES).
NIH Chemical Genomics Center [NCGC]
NIH Molecular Libraries Probe Centers Network [MLPCN]
MLPCN Grant: MH084878
Assay Submitter (PI): Daniel Engel, University of Virgina
Strain 9526-6-2 (MATa his3Delta1 leu2Delta0 lys2Delta0 ura3Delta0 pdr1::KanMX4 pdr3::KanMX4) was a gift from Dan Burke. It was derived by tetrad dissection from two parent strains that had been modified by one step gene replacements. The pdr1::KanMX4 was constructed in BY4741 (MATa his3Delta1 leu2Delta0 met15Delta0 ura3Delta0) and the pdr3::KanMX4 was constructed in BY4742 (MATalpha his3Delta1 leu2Delta0 met15Delta0 ura3Delta0). PCR-mediated one-step gene replacements, matings and tetrad dissections were performed as described previously.[i] The strain 9526-6-2/pYES-NS1 was generated by transformation of 9526-6-2 with the plasmid pYES-NS1 and maintained on synthetic complete medium (SC) lacking uracil. For growth experiments and library screening, a single transformed colony was grown overnight, and the cell number was determined by using a Coulter counter (Beckman Coulter Corp.). The cells were diluted to 5 x 105 cells/mL in SC lacking uracil and containing raffinose and 2% galactose. A portion of this culture was added to preplated test compounds such that the final dimethyl sulfoxide (DMSO) concentration was 1%. Optical density readings at 600 nm (OD600) were taken using a Thermomax microplate reader (Molecular Devices).
Compounds are considered "active" if their OD at 60 hrs in the NS1 strain is >= 0.35; "inconclusive" if < 0.35 and > 0.15; "inactive" if <= 0.15.
"Active" compounds are assigned a score of 90; "inconclusive" a score of 50; "inactive" a score of 10.
Data Table (Concise)