E3 ubiquitin ligases, which bind protein targets, leading to their ubiquitination and subsequent degradation, are attractive drug targets due to their exquisite substrate specificity. However, the development of small-molecule inhibitors has proven extraordinarily challenging as modulation of E3 ligase activities requires the targeting of protein–protein interactions. Using rational design, we have generated the first small molecule targeting the von Hippel–Lindau protein (VHL), the substrate recognition subunit of an E3 ligase, and an important target in cancer, chronic anemia, and ischemia. We have also obtained the crystal structure of VHL bound to our most potent inhibitor, confirming that the compound mimics the binding mode of the transcription factor HIF-1α, a substrate of VHL. These results have the potential to guide future development of improved lead compounds as therapeutics for the treatment of chronic anemia and ischemia.

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.

Amyloid-β precursor protein (APP) is central to the pathogenesis of Alzheimer’s disease, yet its physiological function remains unresolved. Accumulating evidence suggests that APP has a synaptic function mediated by an unidentified receptor for the shed APP ectodomain (sAPP). Here, we showed that the sAPP extension domain directly bound the sushi 1 domain specific to the gamma-aminobutyric acid type B receptor subunit 1a (GABABR1a). sAPP-GABABR1a binding suppressed synaptic transmission and enhanced short-term facilitation in hippocampal synapses via inhibition of synaptic vesicle release. A 17 amino acid peptide corresponding to the GABABR1a binding region within APP suppressed spontaneous neuronal activity in vivo. Our findings identify GABABR1a as a synaptic receptor for sAPP and reveal a physiological role for sAPP in regulating GABABR1a function to modulate synaptic transmission.

SUMMARY Fragment screening is widely used to identify attractive starting points for drug design. However, its potential and limitations to assess the tractability of often challenging protein:protein interfaces have been underexplored. Here, we address this question by means of a systematic deconstruction of lead-like inhibitors of the pVHL:HIF-1α interaction into their component fragments. Using biophysical techniques commonly employed for screening, we could only detect binding of fragments that violate the Rule of Three, are more complex than those typically screened against classical druggable targets, and occupy two adjacent binding subsites at the interface rather than just one. Analyses based on ligand and group lipophilicity efficiency of anchored fragments were applied to dissect the individual subsites and probe for binding hot spots. The implications of our findings for targeting protein interfaces by fragment-based approaches are discussed.

E3 ubiquitin ligases, such as the therapeutically relevant VHL, are challenging targets for traditional medicinal chemistry, as their modulation requires targeting protein-protein interactions. We report novel small-molecule inhibitors of the interaction between VHL and its molecular target HIF1α, a transcription factor involved in oxygen sensing.