Expanding the Structural Diversity at the Phenylene Core of Ligands for the von Hippel-Lindau (VHL) E3 Ubiquitin Ligase: Development of Highly Potent Hypoxia-Inducible Factor-1alpha (HIF-1alpha) Stabilizers

07 March 2023, Version 1
This content is a preprint and has not undergone peer review at the time of posting.


Hypoxia-inducible factor-1alpha (HIF-1alpha) constitutes the principal mediator of cellular adaptation to hypoxia in humans. Hydroxylation of proline residues on HIF-1alpha is recognized by the E3 ligase von Hippel-Lindau (VHL), leading to ubiquitin-dependent proteasomal degradation of HIF-1alpha. In this study, we performed a structure-guided and bioactivity-driven design of new VHL inhibitors with improved cellular activity at blocking HIF-1alpha degradation. With the prototypical ligand VH298 as starting point, our iterative and combinatorial optimization strategy focused on introducing chemical variability into the phenylene unit of the VHL ligand structure and encompassed further points of diversity. The exploitation of tailored phenylene fragments combined with the stereoselective installation of the (S)-configured methyl group at the benzylic position provided VHL ligands with superior binding affinity compared to VH298. Three high-resolution structures of VHL in complex with three new ligands were determined and bioactive conformations of these ligands were explored. The most potent inhibitor (compound 30) exhibited dissociation constants (Kd) lower than 40 nM, independently determined by fluorescence polarization (FP) and surface plasmon resonance (SPR). The improved binding affinity to VHL was accompanied by enhanced cellular potency of 30, considerably exceeding the stabilization of HIF-1alpha by established VHL inhibitors. Our work is anticipated to inspire future efforts towards HIF-1alpha stabilizers and our optimized ligands to serve as an excellent starting point for proteolysis-targeting chimeras (PROTACs) hijacking VHL.


von Hippel-Lindau
E3 ligase
Hypoxia-Inducible Factor
protein-protein interaction
drug design
X-ray crystallography
structure-based drug design

Supplementary materials

Supporting Information
Supporting Figures S1-S6; Supporting Table S1; Supporting Schemes S1-S2; NMR spectra of compounds; Table of X-ray Crystallography data collection and statistics.


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