Biotinylated VCB protein was immobilized onto a streptavidin-functionalized sensor chip allowing real-time measurements of the changes in the refractive index upon binding of ligands to the protein. Our SPR investigations provided double-digit nanomolar values for all selected compounds. As in the FP assay, ligand 30 again possessed the highest affinity to VCB with an SPR-derived Kd value of 25 nM (Figure ). For the eight compounds, second-order rate constants for the association of the binary complexes were between 1.0 × 10 6 M -1 s -1 and 1.7 × 10 6 M -1 s -1 (Table ). The strong affinity of the ligands was reflected by these similarly high association rate constants and dissociation half-lives in a narrow range of 8 to 20 s. Our kinetic data were in line with those of previously investigated, structurally related VHL ligands. A B SPR sensorgrams monitoring real-time interaction of immobilized biotin-VCB protein with 30 (from top to bottom, 1000 nM, 330 nM, 110 nM, 37 nM, 12.3 nM, 4.12 nM, 1.37 nM). Association and dissociation rate constants are listed in Table . Data was fitted to a 1:1 binding model. The binding of ligands to VHL was also examined computationally by molecular docking to refine a 3D structure-based pharmacophore. MIFs using various molecular, e.g. hydrogen bond acceptor/donor and hydrophobic, probes were applied beforehand to identify favorable interaction regions. Following the initial docking setup with two incorporated structural water molecules (Wat406 and Wat450), by a subsequent in-depth examination of the binding pocket, three further structural water molecules (Wat432, Wat440, Wat456) were identified, located at the top of the binding site, which interact with the system via a network of hydrogen bonds (Figure and). Docking poses with compounds 24, 32, and 33 were only slightly different, when two or five structural water molecules were incorporated (Figure ).