Fig. 2. Label-free positive target identification of AVG-233.
(A) Schematic of two RSV L targets examined, representing the full-length polypeptide (top) and a folding-competent (28) truncated L1–1749 polypeptide lacking the MTase and C-terminal (CTD) domains. (B) Purification of polymerase complexes specified in (A) after expression in insect cells. Coomassie blue stain after SDS-PAGE fractionation. (C and D) Dose-dependent BLI-based association (120 s) and dissociation (200 s) curves (left) of AVG-233 with full-length RSV L (C) and L1–1749 (D), with nonlinear fit with one-site specific binding (right). (E) In vitro RdRP assay using a synthetic promoter RNA template for de novo RNA synthesis; representative autoradiogram. (F to H) Primer/template-based in vitro RNA elongation assay, assessing bioactivity and AVG-233 inhibition of L1–1749, with representative autoradiogram (F), densitometric quantitation of elongation products (G), and relative quantitation of each RNA product (H). Symbols represent independent biological repeats (n = 3), and bars represent means. Two-way ANOVA with Dunnett’s post hoc tests.