Figure 4: Live cell imaging of target engagement.
From: In vivo imaging of specific drug–target binding at subcellular resolution
(a) The anisotropy value of Biotin–BODIPY (MW 676.62) increases as a function of binding to NeutrAvidin (MW 60 kDa) (filled triangles), which is suppressed in the presence of 10 × unlabelled biotin as competitor (open triangles). Shown are average±s.d. (n=3); curve fits added for trend visualization. Inset illustration: comparison between the rotation of a free fluorophore in solution and a fluorophore bound to a protein. Owing to the large difference in size of the ligand and the receptor, the increase in FA following binding is large. (b) Average±s.d. anisotropy of non-specifically interacting (green) and PARP bound (red) AZD2281–BODIPY FL (n=3). (c) 3D anisotropy image and corresponding planar and axial cross sections of live HT1080 cells loaded with AZD2281–BODIPY FL. Green corresponds to fluorescent drug molecules that are non-specifically bound. Red corresponds to fluorescent drug molecules with high anisotropy suggesting target (PARP) binding. Normal fluorescence images are shown in Supplementary Fig. 18. Scale bar, 16 μm. (d) 3D anisotropy image and corresponding planar and axial cross sections of live HT1080 cells loaded with AZD2281–BODIPY FL and washed for 30 min. Scale bars, 20 μm.
