E3 ubiquitin ligases are attractive
targets in the ubiquitin–proteasome
system, however, the development of small-molecule ligands has been
rewarded with limited success. The von Hippel–Lindau protein
(pVHL) is the substrate recognition subunit of the VHL E3 ligase that
targets HIF-1α for degradation. We recently reported inhibitors
of the pVHL:HIF-1α interaction, however they exhibited moderate
potency. Herein, we report the design and optimization, guided by
X-ray crystal structures, of a ligand series with nanomolar binding
affinities.
Chemical strategies to using small molecules to stimulate hypoxia inducible factors (HIFs) activity and trigger a hypoxic response under normoxic conditions, such as iron chelators and inhibitors of prolyl hydroxylase domain (PHD) enzymes, have broad-spectrum activities and off-target effects. Here we disclose VH298, a potent VHL inhibitor that stabilizes HIF-α and elicits a hypoxic response via a different mechanism, that is the blockade of the VHL:HIF-α protein–protein interaction downstream of HIF-α hydroxylation by PHD enzymes. We show that VH298 engages with high affinity and specificity with VHL as its only major cellular target, leading to selective on-target accumulation of hydroxylated HIF-α in a concentration- and time-dependent fashion in different cell lines, with subsequent upregulation of HIF-target genes at both mRNA and protein levels. VH298 represents a high-quality chemical probe of the HIF signalling cascade and an attractive starting point to the development of potential new therapeutics targeting hypoxia signalling.
The
von Hippel–Lindau tumor suppressor protein is the substrate
binding subunit of the VHL E3 ubiquitin ligase, which targets hydroxylated
α subunit of hypoxia inducible factors (HIFs) for ubiquitination
and subsequent proteasomal degradation. VHL is a potential target
for treating anemia and ischemic diseases, motivating the development
of inhibitors of the VHL:HIF-α protein–protein interaction.
Additionally, bifunctional proteolysis targeting chimeras (PROTACs)
containing a VHL ligand can hijack the E3 ligase activity to induce
degradation of target proteins. We report the structure-guided design
and group-based optimization of a series of VHL inhibitors with low
nanomolar potencies and improved cellular permeability. Structure–activity
relationships led to the discovery of potent inhibitors 10 and chemical probe VH298, with dissociation constants <100 nM,
which induced marked HIF-1α intracellular stabilization. Our
study provides new chemical tools to probe the VHL-HIF pathways and
new VHL ligands for next-generation PROTACs.
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