Figure 1.
Parallel assay development strategy to interrogate recombinant GCK and GKRP.
Human GCK and GKRP were affinity-purified using GST and FLAG tags, respectively. (A) FRET-based (HTRF) detection. Antibodies recognizing the affinity tags are conjugated to FRET donor and acceptor molecules. Excitation of the donor results in energy transfer (FRET; red dashed oval) to the acceptor only if the acceptor and donor are in close proximity. (B) Reaction scheme for G6PDH/diaphorase dual-coupled assay. The generation of the fluorescent product resorufin (red dashed oval) is measured as the reaction progresses in real time by excitation at 525 nm with emission at 590 nm. (C) Reaction scheme for coupling of ADP generation by GCK to luminescence-based detection. The GCK reaction is allowed to run for a set period of time, and the reaction is then terminated and a two-step reaction utilizes Ultra-Glo™ firefly luciferase to generate bioluminescence (red dashed oval). Reagent 1: ADP-Glo™ Reagent; Reagent 2: Kinase Detection Reagent.
Figure 2.
Optimization and validation of HTRF-based detection of the GCK-GKRP interaction.
(A) Titration of GCK and GKRP. Results are presented as the ratio of the acceptor emission (665 nm) and donor emission (615 nm) for eight different GCK and GKRP concentrations measured at t = 60 minutes. Each data point is mean ± SEM for n = 6. (B) Effect of S6P on HTRF detection of the GCK-GKRP interaction. The experiment was run in the presence of 5 nM GCK and 5 nM GKRP. Values shown are mean ± SEM (n = 8 for each [S6P]). (C–F) Effect of known inhibitors glucose (C), F1P (D), and two GKAs (E–F) on HTRF detection of the GCK-GKRP interaction in the presence of 5 nM GCK, 5 nM GKRP, and 2 µM S6P. Values shown are mean ± SEM (n = 8 for each concentration).
Figure 3.
Quality metrics for the HTRF assay with the LOPAC1280 library in 1536-well plates.
(A) Z’ factor, (B) signal/background, (C) % variance, and (D) calculated IC50 of the control compound GKA-EMD as a function of assay plate.
Figure 4.
Optimization and validation of diaphorase-coupled detection of the GCK enzymatic reaction.
(A) Reaction progress of the dual-coupled diaphorase reaction at various GCK and GKRP concentrations. Each data point is mean ± SEM for n = 8. Dashed lines are NADP+/NADPH controls, with the %NADPH increasing from the bottom (0%) to the top (100%) dashed line. (B) Effect of GKRP modulators on the dual-coupled diaphorase reaction. The reaction included 10 nM GCK and 10 nM GKRP. Results are shown for t = 10 minutes. Each data point is mean ± SEM for n = 2–4.
Figure 5.
Optimization and validation of luminescence-based detection of the GCK enzymatic reaction.
(A) The assay (pH 7.1) contained 5 mM glucose, 0.4 mM ATP, 2 µM S6P, and was terminated after 75 minutes. Each data point is mean ± SEM for n = 4. (B) Effect of GKRP modulators on the luminescence reaction. The reaction included 15 nM GCK and 15 nM GKRP (GCK-GKRP), 4 nM GCK (GCK only), or 0.04 mM ADP and no GCK or GKRP (ADP only). Assays were incubated for 75 minutes. Each data point is mean ± SEM for n = 2–4.
Figure 6.
Localization of GCK in cryopreserved hepatocytes.
Overlay of GCK (FITC channel; green) localization with Hoechst nuclear stain (blue) in cryopreserved hepatocytes from images collected at 10X magnification. The contrast was held fixed for all images, and the full dynamic range of all the 12 bit images has been maintained. (A–C) GCK localization in male Sprague-Dawley rat hepatocytes at (A) 2.5 mM glucose, (B) 16.7 mM glucose, and (C) 2.5 mM glucose and 31.6 µM GKA-EMD. (D–H) GCK localization in cryopreserved human hepatocytes. (D) Translocation of GCK in GKRP-positive cells in two separate lots (TRZ: closed symbols; FOS: open symbols) of human hepatocytes at various concentrations of glucose. (E–F) Translocation of GCK in the presence of GKA-EMD at various concentrations of glucose for donor TRZ and FOS, respectively. (G–H) Representative images from donor TRZ at (G) 2.5 mM glucose and (H) 2.5 mM glucose with 31.6 µM GKA. Data points are mean ± SEM for n = 4–8.