By obstructing the VHL, the powerful, non-toxic, and cell-permeable chemical probe VH-298 causes the hypoxic response. With Kd values of 90 and 80 nM in isothermal titration calorimetry and competitive fluorescence polarization assay, VH-298 is a highly powerful inhibitor of the VHL:HIF-α interaction. VH-298 quickly binds to the VHL complex and takes time to disassociate. When VH-298 is used at a 50 μM concentration, it barely affects more than 100 evaluated cellular kinases, GPCRs, and ion channels in vitro. Cells can pass through VH-298 and it is not hazardous to them. VH-298's permeability is determined to be 19.4 nm s -1 through measurement. VH-298 causes hydroxylated HIF-α to accumulate on-target in a concentration- and time-dependent manner in human cell lines, such as HeLa cancer cells and renal cell carcinoma 4 (RCC4) cells. VH-298 raises EPO mRNA levels by 2.5 times in RCC4-HA-VHL but not in VHL-null RCC4-HA, suggesting that endogenous EPO synthesis can be stimulated by pharmacological inhibition of VHL. While VH-298 and hypoxia both work well to raise PHD2 and HK2 protein levels, in HFF, VH-298 therapy causes a greater increase in BNIP3 protein levels than hypoxia treatment[1].

By obstructing the VHL, the powerful, non-toxic, and cell-permeable chemical probe VH-298 causes the hypoxic response. With Kd values of 90 and 80 nM in isothermal titration calorimetry and competitive fluorescence polarization assay, VH-298 is a highly powerful inhibitor of the VHL:HIF-α interaction. VH-298 quickly binds to the VHL complex and takes time to disassociate. When VH-298 is used at a 50 μM concentration, it barely affects more than 100 evaluated cellular kinases, GPCRs, and ion channels in vitro. Cells can pass through VH-298 and it is not hazardous to them. VH-298's permeability is determined to be 19.4 nm s -1 through measurement. VH-298 causes hydroxylated HIF-α to accumulate on-target in a concentration- and time-dependent manner in human cell lines, such as HeLa cancer cells and renal cell carcinoma 4 (RCC4) cells. VH-298 raises EPO mRNA levels by 2.5 times in RCC4-HA-VHL but not in VHL-null RCC4-HA, suggesting that endogenous EPO synthesis can be stimulated by pharmacological inhibition of VHL. While VH-298 and hypoxia both work well to raise PHD2 and HK2 protein levels, in HFF, VH-298 therapy causes a greater increase in BNIP3 protein levels than hypoxia treatment[1].

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VH298 stabilizes hydroxylated HIF-α subunits (HIF-1α and HIF-2α) in a concentration- and time-dependent manner in various cell lines (e.g., HeLa, HFF, U2OS, RCC4), leading to the upregulation of HIF-target genes (e.g., CA9, GLUT1, PHD2, PHD3, BNIP3, HK2, EPO) at both mRNA and protein levels. [1]
Cellular thermal shift assay (CETSA) demonstrated direct target engagement, showing a significant thermal stabilization of VHL, but not PHD2, in HeLa cell lysates treated with VH298. [1]
Chemoproteomic mass spectrometry confirmed selective capture and displacement of the CRL2VHL complex by a linkable VH298 analogue, supporting its on-target specificity. [1]
VH298 induces HIF-dependent transcriptional activity, as shown by a dose-dependent increase in HRE-luciferase reporter activity and qRT-PCR analysis of HIF-target genes. It is more potent and cell-permeable than its predecessor VH032. [1]
VH298 (up to 150-500 µM) showed negligible cytotoxicity in a range of fibroblast, tumoral, and non-tumoral cell lines, as assessed by CellTiter-Glo viability assays. Its inactive cis-epimer (cisVH298) also showed no toxicity, indicating that potential toxicity is not due to VHL inhibition. [1]

Isothermal Titration Calorimetry (ITC): Experiments were conducted using an ITC200 micro-calorimeter. A solution of VH298 (300 µM) was titrated into a solution containing the VHL-ElonginB:ElonginC (VBC) complex (30 µM). The dissociation constant (Kd) was determined from the binding isotherm. [1]
Competitive Fluorescence Polarization (FP) Assay: Assays were performed in 384-well plates. Each well contained VBC protein (15 nM), a FAM-labeled HIF-1α peptide (10 nM, Kd = 3 nM), and decreasing concentrations of VH298 (two-fold dilutions from 50 µM). Fluorescence polarization was measured (excitation 485 nm, emission 520 nm). IC50 values were determined and Kd values were back-calculated using a displacement binding model. [1]
Surface Plasmon Resonance (SPR): VH298 was dissolved in DMSO and diluted in SPR buffer (20 mM HEPES, 150 mM NaCl, 1 mM DTT, 0.005% Tween 20, pH 7.0) to create a concentration series (31.25 nM to 1 µM, 2% final DMSO). Solutions were injected over a sensor chip with immobilized VBC protein at 10°C. Association (kon) and dissociation (koff) rate constants were determined by fitting the sensorgrams to a 1:1 binding model. [1]

Cell Culture & Treatment: Human cell lines (HFF, HeLa, U2OS, RCC4) were cultured in DMEM supplemented with fetal bovine serum, L-glutamine, and antibiotics. For hypoxia induction, cells were incubated at 1% O2. Cells were treated with VH298 or control compounds (e.g., DMSO, PHD inhibitors IOX2/FG-4592) for specified durations. [1]
Immunoblotting: Cells were lysed in RIPA or Tris lysis buffer containing protease and phosphatase inhibitors. Proteins were resolved by SDS-PAGE, transferred to membranes, and probed with primary antibodies against HIF-1α, HIF-1α-OH (Pro564), HIF-2α, CA9, GLUT1, PHD2, PHD3, BNIP3, HK2, VHL, and β-actin (or SMC1 for CTLs). Detection was performed using HRP-conjugated secondary antibodies and chemiluminescence. [1]
Quantitative Real-Time PCR (qRT-PCR): RNA was extracted from treated cells, reverse transcribed, and analyzed by qRT-PCR using SYBR Green. mRNA levels of HIF-target genes (CA9, GLUT1, PHD2, EPO) were normalized to β-actin or TATA-binding protein (TBP) mRNA. [1]
Cellular Thermal Shift Assay (CETSA): HeLa cell lysates were treated with VH298 (100 µM) or vehicle for 10-30 min at room temperature. Aliquots were heated at different temperatures (40.4-55.4°C) for 3 min, cooled, and centrifuged. Soluble fractions were analyzed by immunoblotting for VHL and PHD2. Band intensities were quantified relative to the lowest temperature control. [1]
Co-immunoprecipitation: HeLa cell lysates (in 1% Triton X-100 buffer) were incubated overnight with an antibody specific for hydroxylated HIF-1α (Pro564) or control IgG. Protein-G-sepharose beads were added to capture immune complexes. After washing, bound proteins were eluted and analyzed by immunoblotting for total HIF-1α. [1]
Parallel Artificial Membrane Permeability Assay (PAMPA): Permeability of VH298 was measured using a pre-coated PAMPA plate. A solution of the compound (10 µM in PBS, pH 7.4) was added to the donor chamber, and PBS was added to the acceptor chamber. After 5 hours at room temperature, compound concentration in both chambers was quantified by UPLC-MS/MS. Effective permeability (Pe) was calculated based on the compound's distribution. [1]
Cytotoxicity Assay: Cells were seeded in 384-well plates and treated with VH298 or its inactive cis-epimer for 24 hours. Cell viability was assessed using the CellTiter-Glo Luminescent Cell Viability Assay, which measures ATP content. Luminescence was measured using a plate reader. For cytotoxic T lymphocytes (CTLs), cell death was assessed by DAPI staining and flow cytometry after 24-hour treatment. [1]

[1]. Potent and selective chemical probe of hypoxic signalling downstream of HIF-α hydroxylation via VHL inhibition. Nat Commun. 2016 Nov 4;7:13312.

VH298 is a high-quality chemical probe that can be used to study the hypoxia signaling pathway by selectively inhibiting the VHL:HIF-α interaction downstream of HIF-α hydroxylation, a mechanism different from that of PHD enzyme inhibitors. It provides a starting point for developing therapies targeting the hypoxia signaling pathway, such as for the treatment of anemia or ischemic diseases. [1] The compound also has potential use as a VHL ligand for the design of PROTAC (proteolytic-targeted chimeras), where its HIF stabilizing activity can be separated from its E3 ligase recruitment function at lower concentrations. [1]

C27H33N5O4S

523.6470

523.225

2097381-85-4

122199236

Off-white to pink solid powder

2.5

3

7

8

37

931

3

S1C([H])=NC(C([H])([H])[H])=C1C1C([H])=C([H])C(=C([H])C=1[H])C([H])([H])N([H])C([C@]1([H])C([H])([H])[C@]([H])(C([H])([H])N1C([C@]([H])(C(C([H])([H])[H])(C([H])([H])[H])C([H])([H])[H])N([H])C(C1(C#N)C([H])([H])C1([H])[H])=O)=O)O[H])=O

NDVQUNZCNAMROD-RZUBCFFCSA-N

InChI=1S/C27H33N5O4S/c1-16-21(37-15-30-16)18-7-5-17(6-8-18)12-29-23(34)20-11-19(33)13-32(20)24(35)22(26(2,3)4)31-25(36)27(14-28)9-10-27/h5-8,15,19-20,22,33H,9-13H2,1-4H3,(H,29,34)(H,31,36)/t19-,20+,22-/m1/s1

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2.5 mg/mL (4.77 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (4.77 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (4.77 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)