Fig. 3
Biochemical and solubility properties of heterocycle-containing tanshinone mimics. (A) REMSA screening of TMs. rM1M2_HuR (3.7 nM) was incubated for 20 min with 1 nM 5ʹ-DY681-labeled RNA probe alone, together with DMSO used as control, or with 2/TM7n, 3–6 and 7b/TM9-13 at 15 µM. The gel was ~ run at 80 V for 100 min. (B) Representative REMSA showing 5/TM11 (0.01–25 µM) dose–response inhibition of the binding between 3.7 nM rM1M2_HuR and 1 nM 5′-DY681-labeled RNA probe. (C) Titration curves of 5/TM11 obtained from REMSA quantification. The calculated half-maximal inhibitory concentration (IC50) is 0.77 µM. (D) Dose–response inhibition by 5/TM11(0.01–100 µM) of the binding between His-tagged rM1M2 HuR protein (20 nM) and 5′-Bi-TNF ARE probe (50 nM), tested in an HTFR assay. The calculated half-maximal inhibitory concentration (IC50) is 0.68 µM; data have been normalized to control (DMSO), and fit nonlinear regression fitting curve according to a one-site binding model in GraphPad Prism. Plots are mean ± s.d. of three independent experiments. (E) Binding of 2/TM7n and 5/TM11 to M1M2 by SPR—representative data from a single measurement. 1:1 binding model fit of steady state responses of 2/TM7n (diamonds, dashed line) and 5/TM11(circles, full line). (F) Solubility of 5/TM11 via LC-MS. The molecule dissolved in DMSO was serially diluted in aqueous medium (phosphate buffer saline, PBS), incubated for 2 h and injected into an LC-MS. Data analysed and plotted with GraphPad Prism as mean ± s.d. of two independent experiments.
