Potent and selective chemical probe of hypoxic signalling downstream of HIF-α hydroxylation via VHL inhibition

Abstract

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.

Structure‐Guided Design of Peptides as Tools to Probe the Protein–Protein Interaction between Cullin‐2 and Elongin BC Substrate Adaptor in Cullin RING E3 Ubiquitin Ligases

“…We hypothesized that Cul2‐derived peptides binding to the EloC site could provide valuable insight on how to target the Cul2‐VBC PPI. Previous work in our research group has led to the development of potent small‐molecule disruptors of the pVHL‐HIF‐1α PPI based on the structure of pVHL bound to a 19‐mer parental peptide derived from HIF‐1α . It was therefore an attractive strategy to explore the potential to apply a similar approach to other non‐HIF binding surfaces on VBC.…”

Proteolysis‐targeting chimeras in drug development: A safety perspective

“…This has been observed at least with the E3 ligases for which natural substrates are known, such as VHL. With VHL based‐PROTACs, several studies have shown that there exists a very large concentration window between PROTAC‐mediated degradation (in the pM–nM range with the best degraders) and stabilization of HIF‐1α, which is only observed at high micromolar range, which is >1000 fold higher than that required for degradation (Bondeson et al, ; Frost et al, ; Maniaci et al, ; Zengerle, Chan, & Ciulli, ). However, the physical and PK properties of PROTACs, which, given their relatively higher MW, are distinct from traditional small‐molecule drugs and achieving appropriate PK will be challenging (Edmondson, Yang, & Fallan, ).…”

“…(2) PROTAC may saturate the proteasome via an increase in target occupancy and stabilization of HIF-1α, which is only observed at high micromolar range, which is >1000 fold higher than that required for degradation (Bondeson et al, 2015;Frost et al, 2016;Maniaci et al, 2017;Zengerle, Chan, & Ciulli, 2015). However, the physical and PK properties of PROTACs, which, given their relatively higher MW, are distinct from traditional small-molecule drugs and achieving appropriate PK will be challenging (Edmondson, Yang, & Fallan, 2019).…”

Recognition of substrate degrons by E3 ubiquitin ligases and modulation by small-molecule mimicry strategies

“…Further optimization of this class of inhibitors led to the discovery of VHL inhibitor VH298 as a novel potent, selective, and cellactive chemical probe of the VHL-HIF pathway [48]. VHL inhibitors have therapeutic potential in certain disease conditions where accumulation of HIF-α subunits and subsequent triggering of hypoxic response could prove beneficial [49].…”