FIGURE 2.

Target verification of direct binding between ALA and AKR1C1. (A) Thermal stabilization of AKR1C1 by ALA (10 μM) in intact NCI-H460 cells was assessed via CETSA analysis. GAPDH was used as control. The melting curves for AKR1C1 and tubulin in NCI-H460 cells in the presence of DMSO (blue curve) and 10 μM ALA (red curve) were plotted by the corresponding mean gray values. All data were normalized to the response observed at each condition at 37°C. (B) Dose-dependent stabilization of AKR1C1 by ALA in intact ALA cells was examined at 52°C by ITDRF analysis. GAPDH was used as control. The dose-response curve was generated by the quantification based on mean gray values. All data were normalized to the intensity at the treatment of 32 μM ALA. (C) SPR bio-sensor was used to detect the binding of ALA to AKR1C1. Apparent K_D value is calculated by SPR data. The fitted K_D is 11.8 μM. (D) The binding mode of ALA to AKR1C1·NADPH as determined by molecular docking. Left, structural overview of docked complex. The protein was shown in a surface representation in light yellow, while ALA and NADPH were shown in a stick representation. Right, zoom-in view of the predicted AKR1C1–ALA interface. The forming hydrogen bond with His-222 was shown as yellow dash. And residues around the predicted binding pocket, including Leu-54, Trp-86, Ile-129, Phe-311, Ile-310, Leu-308, and Trp227, were labeled. (E) The relative catalytic activity of AKR1C1, AKR1C2, and AKR1C3 upon treatment with different concentrations (as indicated) of ALA. Data were presented as mean ± SD (n = 3), **p < 0.01, vs control. Uncropped blots are provided in Supplementary Figure S3.