The six panels show data from experiments on the binding of dual H I F-1 over 2 inhibitors to H I F-1 or H I F-2. Panels A and B display microscale thermophoresis (M S T) data for compounds 1.21 S 9 N and 3.2.16, respectively. Each panel includes a graph of relative fluorescence versus concentration of added compound and a graph of fraction bound to H I F-1 alpha with derived K d estimates. Panels C to F show the results of immunoprecipitation and immunoblot assays for H I F-1 or H I F-2 complex formation inhibition by the same compounds. Each of these panels includes a representative C o-I P image and a graph of the fraction of H I F-1 alpha or H I F-2 alpha bound, normalized to vehicle, with derived I C 50 values. The graphs collectively illustrate the concentration-dependent binding and inhibition effects of the compounds on H I F-1 or H I F-2. All data is approximate.
Biophysical and molecular target engagement of dual HIFi . (A and B) MST of fluorophore-tagged recombinant human HIF-1α following the addition of 1.21S9N (A) or 3.2.16 (B) was performed. Data are presented as relative fluorescence at each concentration of added compound (nM; upper panels) and fraction bound with derived Kd estimates (mean ± SD, n = 4; lower panels). (C–F) Lysates of Hep3B cells with OE of HIF-1α or HIF-2α were treated with HIFi and then incubated with lysate of cells with OE of HIF-1β. Concentration-dependent inhibition of HIF-1α–HIF-1β (C and E) or HIF-2α–HIF-1β (D and F) complex formation by 1.21S9N (C and D) or 3.2.16 (E and F) was analyzed by immunoprecipitation for HIF-α and immunoblot assay for HIF-1β. Representative Co-IP (upper panels) and fraction of HIF-1β bound (normalized to vehicle) with derived IC50 values (mean ± SD, n = 3; lower panels) are shown. OE, overexpression. Source data are available for this figure: SourceData F2.