Design of orthogonal molecular probes targeting engineered von-Hippel Lindau (VHL) E3 ubiquitin ligase for the control of intracellular protein levels

Project Information

orthoPROTACS

Grant agreement ID: 328030

Closed project

Start date 8 April 2013

End date 7 April 2015

Funded under

• Specific programme "People" implementing the Seventh Framework Programme of the European Community for research, technological development and demonstration activities (2007 to 2013)

Total cost € 221 606,40

EU contribution € 221 606,40

Coordinated by

UNIVERSITY OF DUNDEE
United Kingdom

Final Report Summary - ORTHOPROTACS (Design of orthogonal molecular probes targeting engineered von-Hippel Lindau (VHL) E3 ubiquitin ligase for the control of intracellular protein levels)

Drugging the von Hippel-Lindau (VHL) E3 ligase

Carles Galdeano, Alessio Ciulli

Division of Biological Chemistry and Drug Discovery, College of Life Sciences, University of Dundee, Dow Street, Dundee, DD1 5EH, Scotland, UK.

During normal cellular homeostasis, proteins are constantly synthesized and destroyed. The most common degradation pathway for proteins is the ubiquitin-proteasome system (UPS), a highly regulated signaling cascade that is responsible for the controlled degradation of a large number of proteins upon their polyubiquitination and ultimately hydrolysis by the proteasome. Key to protein degradation by the UPS is the recruitment of the substrate protein by an E3 ubiquitin ligase. Ubiquitin ligases confer substrate specificity for ubiquitination and could provide more attractive targets for therapeutic intervention than current proteasome inhibitors, making this unconventional enzyme class most appealing for drug discovery efforts. To date however, the development of small molecules against E3 ligases has been rewarded with limited success, in part because modulating their activity and regulation requires the targeting of protein-protein interactions.

One E3 ubiquitin ligase with important biological relevance where some success has recently been made is the von Hippel-Lindau (VHL) E3 ligase. The primary substrate of VHL is the Hypoxia Inducible Factor 1α (HIF-1α), a transcription factor that regulates over 2% of human genes, particularly those related to oxygen sensing and the hypoxic response. However, under normal oxygen levels HIF-1α is constitutively expressed and targeted for proteasomal degradation upon hydroxylation by prolyl hydroxylases domain (PHD) leading to specific recruitment by VHL and subsequent VHL-mediated polyubiquination. Small molecule inhibition of this pathway would be expected to mimic the physiological response to low oxygen levels by increasing the expression of genes involved with the hypoxic response.

Development of the first nanomolar inhibitors of the VHL:HIF-1α protein:protein interaction

A series of ligands were reported by the Ciulli group and collaborators as small-molecule inhibitors of the VHL:HIF-1α interaction, however these molecules bound to VHL with moderate potency, the best ones still only in the single-digit micromolar range.[1,2] During the duration of this Marie-Curie Intra-European Fellowship, we have designed, synthetised and optimised the next-generation of small molecules targeting VHL, with binding affinities in the nanomolar range and improved lipophilicity. X-ray crystal structures and ITC studies revealed new interactions and have elucidated the structural and thermodynamic determinants for the improvement in binding affinities, thus providing crucial information for future ligand optimization campaigns. Our best ligand at this point provided an excellent starting point to validate VHL as a drug target.

Validating with small-molecules the VHL:HIF-1α protein:protein interaction as a new target in the hypoxia pathway

Althought nanomolar inhibitors of the VHL:HIF-1α have been reported in the context of this Marie Curie Project,[3] the functional and biological consequences of inhibiting this interaction inside cells have remained elusive. For that, we have demonstrated that these ligands are cell-penetrant and able to induce dose-dependent stabilization of HIF-1α in different cell types.[4] We have also showed using chemoproteomics the exquisite selectivity of our VHL ligands. Guided by our crystal structures of VHL, we have designed a new even more potent VHL:HIF-1α inhibitor in the last period of the Project, the most potent compound described to date (Kd = 40 nM) which consistently shows cellular activity and achieves strong target engagement in a CETSA assay[5] in cells lysates. The compound-stabilized hydroxylated HIF-1α is transcriptionally active, as demonstrated by HRE-Luciferase assays and qRT-PCR. Free intracellular concentrations of ligands have been measured to enlight the mismatches that we have been observed between the biophysical in vitro affinities and the cellular activities.

Conclusions

In conclusion, we have demonstrated that potent and selective VHL compounds trigger a functional dose-dependent response downstream of HIF-1α hydroxylation in the hypoxia signaling cascade pathway inside cells. Our study establishes a blueprint for biochemical characterization of VHL:HIF-1α inhibitors as high-quality chemical probes to drive a HIF dependent hypoxic response. These inhibitors could help to elucidate new roles of both VHL and HIF in physiological and pathophysiological conditions, and will motivate future drug development efforts at validating the VHL:HIF-1α protein-protein interaction as a target in relevant animal models in vivo.

References

1. Buckley, D. L. et al. Targeting the von Hippel-Lindau E3 ubiquitin ligase using small molecules to disrupt the VHL/HIF-1α interaction. J. Am. Chem. Soc. 134, 4465–4468 (2012).
2. Dias, D. M.; Van Molle, I.; Baud, M. G. J.; Galdeano, C.; Geraldes, C. F. G. C.; Ciulli, A. Is NMR Fragment Screening Fine-Tuned to Assess Druggability of Protein–Protein Interactions? ACS Med. Chem. Lett. 5, 23-28 (2014).
3. Galdeano, C. et al. Structure-guided design and optimization of small molecules targeting the protein-protein interaction between the von Hippel-Lindau (VHL) E3 ubiquitin ligase and the hypoxia inducible factor (HIF) alpha subunit with in vitro nanomolar affinities. J. Med. Chem. 57, 8657–8663 (2014).
4. Galdeano, C.; Julianty, J.; Shimamura, S.; Soares, P.; Gadd, M.S.; Epemolu, O.; Grzes, K.; Cantrell, D.A.; Read, K.D. Rocha, S.; Bantscheff, M.; Grandi, P.; Ciulli, A. Stabilizing transcriptionally active hypoxia-inducible factor-1 α (HIF-1α) with chemical probes that disrupt the VHL:HIF-1α protein-protein interaction. Under revision.
5. Martinez Molina, D. et al. Monitoring drug target engagement in cells and tissues using the cellular thermal shift assay. Science 341, 84–87 (2013).

Related documents

• final1-figure1-fr.pdf
• final1-figure2-fr.pdf