Computational discovery of HIF-1α/VHL protein-protein interaction inhibitors for hypoxic cell protection

The HIF-1α/VHL protein-protein interaction regulates cellular hypoxic adaptation. Targeting this PPI offers therapeutic potential for ischemia, yet the structural diversity and direct cytoprotective applications of reported VHL ligands remain limited. Here, we developed a computational virtual-screening workflow informed by deep learning-based interface analysis. DDMut-PPI was used to nominate putative auxiliary interface residues for construction of an alternative pharmacophore model. Screening of 24,893 molecules followed by fluorescence-polarization testing identified four candidates with IC₅₀ values below 10 μM. Cmpd16 (CAS 1072833-77-2; ixazomib) showed the highest measured affinity in this panel (IC₅₀ = 0.41 μM). Cmpd16 showed no detectable loss of viability under the reported assay conditions and produced a VHL-dependent pattern of HIF-1α and hydroxylated HIF-1α stabilization. In an oxygen-glucose deprivation/reoxygenation model, 10 μM Cmpd16 improved endothelial-cell migration and tube formation and was associated with increased VEGF and GLUT1, reduced ROS accumulation, and reduced cleaved caspase-3. Three independently initialized 200-ns Desmond production simulations showed recurring Pro99 and His110 contacts but replica-dependent protein and ligand dynamics, supporting a computationally plausible rather than unique Cmpd16 orientation. The comparison between the two selected ten-compound panels remained exploratory (two-sided Fisher's exact test, p = 0.0867), and residue causality requires experimental mutagenesis.