(A) Interaction of GNF2-biotin with a recombinant, soluble form of the DENV2 E protein in its prefusion conformation occurs in a concentration-dependent manner detectable in a bead-based proximity assay (AlphaScreen). (B) Concentration-dependent disruption of this interaction by representative small molecule inhibitors of DENV2 infectivity is correlated with antiviral potency. Compound 3m is a negative control compound that exhibits no activity in the DENV2 infectivity assay (Table S1). IC90 values in the infectivity assay are GNF-2, 18 μM and 3-110- 22, 0.77 μM (Schmidt, et al., 2012). (C) Structure of GNF2-FITC. (D) Interaction of GNF2- FITC with prefusion sE is detectable by increasing anisotropy in a fluorescence polarization assay. No interaction of GNF2-FITC with a soluble form of the DENV2 E protein in the post- fusion, trimeric conformation was detected. Representative data for two or more independent experiments are shown. Data represent the mean ± standard deviation for a representative experiment performed n ≥ 2.