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

Galdeano, Carles; Gadd, M.S.; Soares, Pedro; Scaffidi, Salvatore; Molle, Inge Van; Birced, Ipek; Hewitt, Sarah; Dias, David M.; Ciulli, A.

doi:10.1021/jm5011258

Cited by 299 publications

(354 citation statements)

References 22 publications

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“…Furthermore, the weak hot spots on the two sides of the L-Hyp moiety also contribute to binding. In fact, optimization of 7 resulted in several inhibitors with high nanomolar affinities (37), all retaining L-Hyp as their anchor.…”

Section: Resultsmentioning

confidence: 99%

Ligand deconstruction: Why some fragment binding positions are conserved and others are not

Kozakov

Hall

Jehle

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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Fragment-based drug discovery (FBDD) relies on the premise that the fragment binding mode will be conserved on subsequent expansion to a larger ligand. However, no general condition has been established to explain when fragment binding modes will be conserved. We show that a remarkably simple condition can be developed in terms of how fragments coincide with binding energy hot spots—regions of the protein where interactions with a ligand contribute substantial binding free energy—the locations of which can easily be determined computationally. Because a substantial fraction of the free energy of ligand binding comes from interacting with the residues in the energetically most important hot spot, a ligand moiety that sufficiently overlaps with this region will retain its location even when other parts of the ligand are removed. This hypothesis is supported by eight case studies. The condition helps identify whether a protein is suitable for FBDD, predicts the size of fragments required for screening, and determines whether a fragment hit can be extended into a higher affinity ligand. Our results show that ligand binding sites can usefully be thought of in terms of an anchor site, which is the top-ranked hot spot and dominates the free energy of binding, surrounded by a number of weaker satellite sites that confer improved affinity and selectivity for a particular ligand and that it is the intrinsic binding potential of the protein surface that determines whether it can serve as a robust binding site for a suitably optimized ligand.

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Section: Resultsmentioning

confidence: 99%

Ligand deconstruction: Why some fragment binding positions are conserved and others are not

Kozakov

Hall

Jehle

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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“…1). We therefore designed VH298, based on our crystal structure of inhibitor VH032 bound to VHL30. VH298 bears a cyanocyclopropyl group in place of the terminal methyl group of VH032 (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…VH298 retains the same overall binding mode of VH032 (ref. 30). The cyanocyclopropyl group fits snugly at the far left-hand side pocket of the HIF-1α binding site on VHL, with the cyclopropyl moiety projecting towards the aliphatic side chain of Arg69, and the cyano group pointing away from the surface and towards solvent in an anti conformation relative to the amide carbonyl.…”

Section: Resultsmentioning

confidence: 99%

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

et al. 2016

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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.

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“…Much of the focus to date has centered on disrupting the VHL–HIF-1α interface using small molecules (Buckley et al, 2012; Galdeano et al, 2014; Van Molle et al, 2012). The targeting of the VHL–Cul2 or EloC–Cul2 interaction to prevent HIF downregulation has not yet been explored.…”

Section: Discussionmentioning

confidence: 99%

Insights into Cullin-RING E3 Ubiquitin Ligase Recruitment: Structure of the VHL-EloBC-Cul2 Complex

et al. 2015

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Summary The von Hippel-Lindau tumor suppressor protein (VHL) recruits a Cullin 2 (Cul2) E3 ubiquitin ligase to downregulate HIF-1α, an essential transcription factor for the hypoxia response. Mutations in VHL lead to VHL disease and renal cell carcinomas. Inhibition of this pathway to upregulate erythropoietin production is a promising new therapy to treat ischemia and chronic anemia. Here we report the crystal structure of VHL bound to a Cul2 N-terminal domain, Elongin B (EloB), and Elongin C (EloC). Cul2 interacts with both the VHL BC box and cullin box and a novel EloC site. Comparison to other cullin E3 ligase structures shows that there is a conserved, yet flexible, cullin recognition module and that cullin selectivity is influenced by distinct electrostatic interactions. Our structure provides a structural basis for the study of the pathogenesis of VHL disease and the rationale design of novel compounds that may modulate cullin–substrate receptor interactions.

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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

Cited by 299 publications

References 22 publications

Ligand deconstruction: Why some fragment binding positions are conserved and others are not

Ligand deconstruction: Why some fragment binding positions are conserved and others are not

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

Insights into Cullin-RING E3 Ubiquitin Ligase Recruitment: Structure of the VHL-EloBC-Cul2 Complex

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