FIG 4.

Compound target identification. (A) Surface biotinylation of BSR T7/5 cells transiently transfected with expression plasmids encoding IAV-WSN HA and incubated in the presence of 10 μM compound or an equivalent volume of vehicle (DMSO). Streptavidin-precipitated surface proteins were subjected to SDS-PAGE, and blots were decorated with specific antibodies directed against HA or the host cell transferrin receptor (TfR), as an internal standard. Bars show average results and SD for densitometric quantitation of signal intensities of three independent repeats. Control cells received equal amounts of empty vector DNA (mock). One-way ANOVA with Sidak's multiple-comparison posttest was used for statistical analysis (ns, not significant [P ≥ 0.05]). (B) Purification of foldon-stabilized soluble IAV-WSN HA protein. Nickel affinity chromatography-purified soluble HA was biotinylated in vitro at a C-terminally inserted Avitag prior to SDS-PAGE. Gels were either subjected to Western blotting (WB) using specific antibodies directed against IAV HA or HRP-conjugated streptavidin or directly subjected to silver staining (Ag stain). (C) BLI analysis of compound binding to purified soluble IAV-WSN HA protein immobilized on high-density streptavidin-coated sensors. Association and dissociation curves are shown for all hit compounds and an inactive analog of GRP-71271 at different concentrations. Lines represent regression modeling, and numbers represent dissociation values (K_D) and the goodness of fit of the underlying regression model (R²).