Determination of the HIF-1α stabilization by VHL inhibitors. Next, it was intended to confirm the VHL-inhibiting activity of selected ligands in a cellular environment. All of the tested 14 ligands had an FP-derived Kd value lower than 200 nM. The corresponding assay is predicated on the blockade of the HIF-1α-OH binding site of VHL by a synthetic ligand, at which successful competition with endogenous HIF-1α-OH protein prevents ubiquitination and hence leads to an accumulation of HIF-1α-OH. The ability of inhibitors to stabilize HIF-1α was inspected both in HeLa and HEK 293 cells by detecting HIF-1α protein levels by means of Western blotting analysis (Figure ). The experiment involved treating cells at an inhibitor concentration of 50 µM for 2 hours, these appropriate conditions were adapted from previous studies with related VHL inhibitors. We used primary antibodies against HIF-1α and HIF-1α-OH (Pro564). The quantified results of the immunoblotting experiments (Table ) were normalized to reference compound 1 (VH298). The HIF-1α antibody used for immunoblotting is unspecific regarding Pro564 hydroxylation and will bind to both HIF-1α and HIF-1α-OH. As the cells were handled under normoxic conditions, Pro564 hydroxylation will continue to take place, generating HIF-1α-OH, which gets accumulated due to blockage of its binding site at VHL. The HIF-1α-OH antibody, on the other hand, specifically interacts only with HIF-1α-OH hydroxylated at Pro564. All investigated compounds exhibited HIF-1α stabilization activity compared to the DMSO control (Table ). Unambiguous SARs could be deduced, which were consonant with the results from our FP and SPR studies. The most promising candidate, ligand 30, also caused a maximum enhancement of HIF-1α and HIF-1α-OH protein levels in both HeLa and HEK 293 cells. The three most potent HIF stabilizers, i.e. 30, 32, and 33, all exhibiting a comparatively high logD value of 2.5, outperformed reference compound 1 considerably (Figure , Table ), indicating their outstanding cellular activity. Since the applied anti-HIF-1α antibody does not discriminate between the hydroxylated and non-hydroxylated HIF-1α species, the immunoblotting data reflected the increased overall HIF-1α abundance. When employing a specific anti-HIF-1α-OH (Pro564) antibody, congruent data were obtained (Figure , Table ). The increase in the HIF-1α-OH level can be attributed to two interconnected events, (i) the inhibition of VHL-catalyzed ubiquitination and subsequent proteasomal degradation, and (ii) the under normoxic conditions ongoing generation of HIF-1α-OH by PHD, the activity of which is controlled by a complex regulation. As noted in a previous report, the phenylene-unsubstituted compound 38 (VH101) behaved as a superior VHL inhibitor. The cellular activity of this benchmark compound was considered for reasons of comparability. Encouragingly, compound 30 was shown to be approximately 2-fold more active than 38, both in HeLa and HEK 293 cells, and with respect to HIF-1α and HIF-1α-OH stabilization. VHL inhibitor 30 represents the most potent lowmolecular-weight HIF-1α stabilizer known so far. Generally, compounds' overall cell permeability influences their capability for HIF-1α stabilization. However, in most of our inhibitors, binding affinity and cellular potency correlated well, thus suggesting permeability was not substantially altered by the subtle structural changes.