Figure 3: Measuring the intracellular residence time of HDAC inhibitors at HDAC1.
From: Target engagement and drug residence time can be observed in living cells with BRET
(a) Structure of the inhibitor FK228 in the prodrug form (left) and the form activated by intracellular reduction (right). (b) Illustration of assay method for measuring intracellular residence time using BRET. Live cells expressing the target protein fused to Nluc are equilibrated with a near-saturating concentration of compound. The cells are then washed to remove unbound compound and treated with a near-saturating concentration of a tracer. The profile of compound with slow dissociation kinetics from the target impedes tracer binding, which slows production of the BRET signal. (c) Residence time analysis by BRET reveals remarkably slow dissociation rates for FK228 (red) and TDP-A (blue), compared with mocetinostat (green), SAHA (grey) or vehicle/DMSO control (black). Data are normalized to maximum signal (vehicle/DMSO-treated) versus full-occupancy control (10 μM SAHA, no washout). The kinetic traces for FK228 and TDP-A are nearly indistinguishable from the full occupancy control (10 μM SAHA, no washout). Data are mean±s.e.m.. of four independent experiments.
