VH298

Reagent Authentication Certificate:

Chemical Probes Portal VH298.pdf

Protein target name:

VHL

Mechanism of action:

Protein-protein interaction inhibitor

Recommended Concentration for use in cells:

10-100 μM

Probe Availability

Tocris (VH 298)

Probe Information

Target and Probe Information

Target Details

Target class:

VHL, VHL1, HRCA1, RCA11, pVHL, von Hippel-Lindau tumor suppressor, E3 ubiquitin protein ligase

Probe Details

Chemical Probes Portal ID:

CPP769

PubChem CID:

122199236

SMILES string:

O=C(N[C@@H](C(C)(C)C)C(N1C[C@@H](C[C@H]1C(NCC2=CC=C(C3=C(N=CS3)C)C=C2)=O)O)=O)C4(C#N)CC4

InChi Key:

GYJMGEJLYDLYLJ-IUVJQGSXSA-N

Control compound:

cisVH298

Orthogonal probe:

CM11

Physico-chemical properties

Solubility - preferred solvent:

DMSO

Solubility - concentration:

Soluble in DMSO at concentrations >100 mM

Potential reactivities:

Not known

NMR:

1H NMR (CDCl3, 400 MHz): δ 8.68 (s, 1H), 7.36 (q, 4H, J = 16.0, 8.0 Hz), 6.96 (d, 1H, J = 8.0 Hz), 4.75 (t, 1H, J = 8.0 Hz), 4.61-4.56 (m, 2H), 4.42 (d, 1H, J = 8.0 Hz), 4.34 (dd, 1H, J = 16.0, 8.0 Hz), 3.95(d, 1H, J = 8.0 Hz), 3.62 (dd, 1H, J = 16.0, 8.0 Hz), 2.65-2.58 (m, 1H), 2.53 (s, 3H), 2.13-2.08 (m, 1H), 1.71- 1.58 (m, 4H), 0.96 (s, 9H); 13C NMR (CDCl3, 101 MHz): δ 171.1, 170.4, 166.2, 150.5, 148.7, 138.0, 131.7, 131.3, 129.8, 128.4, 119.7, 70.4, 58.8, 58.5, 56.6, 43.5, 35.8, 35.2, 26.5, 18.1, 16.2, 13.8. HRMS (ESI) m/z: [M++1] calculated for C27H34N5O4S: 524.2331; observed: 524.2344.

Storage Recommendations

Storage (dry powder):

Freezer (-20ºC). No degradation detected >1 year.

Storage (solution):

Freezer (-80ºC). No degradation detected after >2 months.

Storage sensitivities:

Not known

Validation in Cells and Model Organisms

In vitro validation

On target activity

Potency:

90 nM

Potency assay:

In isothermal calorimetry assays, VH298 bound VBC with a KD of 90 nM; in flourescence polarization assays using a HIF-1alpha peptide, VH298 bound VBC with a KD of 80 nM. Using SPR, VH298 had a Kon = 6.47 X 10^5/M*sec and a Koff = 0.065/sec.

PDB ID for probe-target interaction (3D structure):

5LLI

Structure-activity relationship:

See Galdeano et al., J. Med. Chem. 2014 (doi: 10.1021/jm5011258)

Off target activity

Probe Selectivity in Vitro:

In Dundee MRC-PPU Express Screen of 50 kinases, VH298 did not substantially inhibit any kinases at 50 uM. In CEREP ExpressProfile assay of 50 GPCRs and ion channels, VH298 did not bind any targets at 50 uM.

In cell validation

On target activity in cells

Potency in cells:

Not done

Potency assay (cells):

In HeLa cells, VH298 led to the concentration-dependent accumulation of HIF-1alpha (hydroxylated form) and HIF-2alpha.

Target engagement assay (cells):

In CETSA, VH298 stabilized VHL.

Off target activity in cells

Probe Selectivity in Cell:

In chemoproteomic MS assays using an immobilized, linkable analogue of VH298, RBX1, CUL2, VHL, and TCEB2 were the only proteins that were reproducibly detected in a dose-dependent competition experiment.

Toxicity

Cytoxicity assay:

Yes

Notes on cytotoxicity:

In several cell types (fibroblasts, tumor cell lines and non-tumor cell lines) at 150 uM, VH298 was not cytotoxic.

Primary Reference

Reference (doi):

DOI: 10.1038/ncomms13312

Reference (PMID):

27811928

Endorsed probe

The Chemical Probes Portal endorses this compound as chemical probe for use in cells.

SAB Rating

Rating for use in Cells

3 review(s)

Rating for use in Model Organisms

no rating

SAB Members

SAB Comments

Aqueous solubility data should be provided for this probe.

Dec 29 2016 - 4:41pm

A useful probe that blocks VHL:HIF-a interaction.

Dec 30 2016 - 1:13pm

A selective probe that inhibits the VHL-HIFa interaction. There is a large difference between FP, ITC, and SPR-determined affinity and cell-based activity (recommended for use at 10-100 uM). I recommend performing isothermal dose response CETSA to assess affinity for VHL in cells.

Jan 4 2017 - 11:37am

Author note: Regarding "Aqueous solubility data should be provided for this probe."

We have now measured the solubility of this probe. VH298 was soluble at 250 uM in water (unbuffered solution) in the presence of 2.5% v/v DMSO.

Apr 7 2017 - 4:48pm

The probe is soluble in DMSO at concentrations >100 mM.

Apr 28 2017 - 9:58pm

VH298

Probe Availability

Probe Information

Target Details

VHL, VHL1, HRCA1, RCA11, pVHL, von Hippel-Lindau tumor suppressor, E3 ubiquitin protein ligase

Probe Details

Physico-chemical properties

In vitro validation

In cell validation

Toxicity

Primary Reference

Supporting Organizations

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VH298

Probe Availability

Probe Information

Target Details

VHL, VHL1, HRCA1, RCA11, pVHL, von Hippel-Lindau tumor suppressor, E3 ubiquitin protein ligase

Probe Details

Physico-chemical properties

In vitro validation

In cell validation

Toxicity

Primary Reference

Supporting Organizations

Interested in supporting the portal?