PTEN (IC50 = 35 nM)
PTEN (phosphatase and tensin homologue deleted on chromosome 10).
Inhibition constants (with OMFP as substrate): Kic = 27 ± 6 nM, Kiu = 45 ± 11 nM (noncompetitive inhibition). [1]
With PIP3 as substrate: Kic = 17 ± 8 nM, Kiu = 74 ± 31 nM (n=2). [1]
IC50 (with OMFP as substrate): 46 ± 10 nM. [1]
Previously determined IC50 with PIP3 as substrate: 35 ± 2 nM. [1]

Since VO-OHpic is a sterically demanding compound with two OHpic and one oxo ligand, it makes sense that steric hindrance from the bound substrate would impact the inhibitor's subsequent binding. At low nanomolar doses (IC50, 46±10 nM), VO-OHpic substantially reduced PTEN activity, which is consistent with the efficacy found in a PIP3-based assay earlier (IC50, 35±2 nM). It was found that the inhibitory constants Kic and Kiu were, respectively, 27±6 and 45±11 nM[1]. A promising selective and effective PTEN inhibitor is VO-OHpic. The most effective PTEN lipid phosphatase activity inhibitor is VO-OHpic (IC50=35 nM)[2].

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VO-OHpic potently inhibits recombinant PTEN in a reversible manner. The inhibition is noncompetitive with respect to both the artificial substrate OMFP and the physiological substrate PIP3, as demonstrated by an increase in Km and a decrease in Vmax with increasing inhibitor concentration. The inhibition constants Kic and Kiu were determined to be 27 ± 6 nM and 45 ± 11 nM respectively using OMFP as substrate, and 17 ± 8 nM and 74 ± 31 nM using PIP3 as substrate. The IC50 of VO-OHpic for PTEN was 46 ± 10 nM with OMFP substrate, consistent with the previously reported value of 35 ± 2 nM using PIP3. VO-OHpic has high selectivity for PTEN over other PTPs (which are inhibited only at high micromolar concentrations). [1]

Mice's PTEN was suppressed by an intraperitoneal injection of VO-OHpic (10 μg/kg) 30 minutes before to ischemia, followed by exposure to ischemia for 30 minutes and reperfusion for 120 minutes. Triphenyltetrazolium chloride (TTC) was used to measure the size of the myocardial infarct at the conclusion of the experiment. In mice treated with VO, the size of the myocardial infarct was considerably smaller (25±6% vs. 56±5%, n=7, P<0.01). Between the two groups, there was no difference in the risk area (46±3% vs. 57±3%, n=7, P>0.05) [3].

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In mice, intraperitoneal injection of VO-OHpic (10 μg/kg) 30 minutes before ischemia significantly increased Akt phosphorylation in the myocardium (145 ± 10 AU vs. 76 ± 8 AU in control, n=4, P<0.01). [3]
VO-OHpic treatment decreased left ventricular systolic pressure (60 ± 3 vs. 70 ± 2 mmHg, n=7, P<0.01), heart rate (349 ± 12 vs. 427 ± 27 beats/min, n=7, P<0.01), and +dp/dt_m (4061 ± 215 vs. 4818 ± 316 mmHg/s, n=7, P<0.05) before ischemia. [3]
After 30 minutes of ischemia followed by 120 minutes of reperfusion, VO-OHpic-treated mice showed better cardiac functional recovery: at the end of reperfusion, left ventricular systolic pressure was higher (65 ± 4 vs. 53 ± 4 mmHg, n=7, P<0.05), heart rate was higher (476 ± 26 vs. 381 ± 19 beats/min, n=7, P<0.05), +dp/dt_m was higher (4432 ± 198 vs. 2765 ± 333 mmHg/s, n=7, P<0.01), -dp/dt_m was higher (3699 ± 331 vs. 1921 ± 401 mmHg/s, n=7, P<0.01), and left ventricular end-diastolic pressure was lower (-6 ± 2 vs. 2 ± 3 mmHg, n=7, P<0.05) compared to control. [3]
VO-OHpic significantly decreased myocardial infarct size after ischemia-reperfusion (25 ± 6% vs. 56 ± 5% of area at risk, n=7, P<0.01), with no difference in the area at risk between groups (46 ± 3% vs. 57 ± 3%, n=7, P>0.05). [3]

PTEN assay with OMFP as substrate: OMFP cyclohexylammonium salt was dissolved in DMSO to 20 mM and further diluted with 1% DMSO to test concentrations. Assays were performed in 100 mM Tris (pH 7.4) containing 2 mM DTT at room temperature (20°C). Reactions were initiated by adding OMFP to the PTEN buffer mixture. Hydrolysis of OMFP to OMF was monitored by measuring fluorescence change in a 96-well microtiter plate (excitation 485 nm, emission 525 nm). DMSO was used as solvent to allow a 20 mM stock solution; the final solvent concentration in the assay was kept low. For high OMFP concentrations (>1 mM), inner filter effects were verified by dilution and corrected if present. [1]
PTEN assay with PIP3 as substrate: Enzyme activity was measured in 100 mM Tris (pH 7.4) containing 2 mM DTT. PIP3 (diC16 sodium salt) was dissolved in distilled water to 1 mM and further diluted with water to test concentrations. The assay was performed at 30°C for 20 minutes. The reaction was stopped by adding 2.25 volumes of colour reagent (5 mM malachite green, 17 mM ammonium heptamolybdate, 77 mM bismuth citrate, 1.7 M HCl). The mixture was developed for 10 minutes, and absorbance was read at 650 nm. [1]
PTEN activity assay in the presence of VO-OHpic: VO-OHpic was dissolved in DMSO (100 μM) and diluted further to required concentration with 1% DMSO. For inhibition studies, PTEN was preincubated with VO-OHpic at room temperature for 10 minutes before substrate addition to initiate the reaction. Background absorbance (malachite green assay) and fluorescence (OMFP assay) were determined with VO-OHpic in assay buffer and corrected in data analysis. [1]
Reversibility of VO-OHpic inhibition - inhibitor dilution assay: PTEN was preincubated with a high concentration of inhibitor (300 nM) and then diluted by adding reaction buffer with no inhibitor present. The remaining PTEN activity towards OMFP was measured and compared to controls. Four experiments were performed: (1) PTEN activity without inhibitor; (2) PTEN activity with 30 nM VO-OHpic; (3) PTEN preincubated with 300 nM VO-OHpic then diluted 10-fold to 30 nM; (4) PTEN preincubated with 300 nM VO-OHpic then diluted with buffer containing 300 nM VO-OHpic. Activity recovery after dilution indicated reversible inhibition. [1]

Cell culture: NIH 3T3 fibroblasts, 3T3-L1 fibroblasts (or differentiated to adipocytes), and UmUc-3 human urinary bladder carcinoma cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% newborn calf serum at 37°C with 5% CO₂.
Western blotting: Quiescent cells were prestimulated with low-dose insulin (0.2 μg mL⁻¹) for 5 minutes, then treated with indicated concentrations of VO-OHpic for 15 minutes. For comparison, full stimulation was with 10 μg mL⁻¹ insulin. Cells were lysed in SDS sample buffer. Proteins were separated by SDS-PAGE and blotted onto PVDF membrane. Membranes were probed with mouse monoclonal anti-phospho-Akt (Ser473) or anti-phospho-Akt (Thr308) antibodies, followed by HRP-conjugated anti-mouse antibody. Stripped membranes were reprobed with rabbit anti-Akt antibody and HRP-conjugated anti-rabbit antibody for loading control.
Immunofluorescence for Akt: Cells on poly-1-lysine coated coverslips were starved overnight, preincubated with inhibitors and stimulated as indicated. Cells were fixed in 4% paraformaldehyde (PFA), quenched, permeabilized with Triton X-100, and blocked. Cells were incubated with fluorescently labeled anti-phospho-Akt (Ser473) antibody, washed, and counterstained with DAPI. Coverslips were mounted and observed under a fluorescence microscope.
Detection of PtdIns(3,4,5)P3 and PtdIns(3,4)P2: Fixed cells were probed with a recombinant GST-tagged PH domain of Akt/PKB (amino acids 5-108), which was detected with an Alexa Fluor labeled anti-GST antibody. The staining pattern was visualized by fluorescence microscopy.
Immunofluorescence for FoxO3a: Cells were treated as indicated, fixed, permeabilized, and stained with a primary anti-FoxO3a antibody and a secondary Cy5-labeled antibody. Nuclei were stained with DAPI.
Glucose uptake assay: Differentiated L1 adipocytes on coverslips were serum- and glucose-starved for 4 hours. After preincubation with VO-OHpic and insulin, cells were treated with a fluorescent glucose analogue (2-NBDG) for 10 minutes. A negative control with D-glucose was included. Coverslips were washed, counterstained with DAPI, and imaged.
Transfection and luciferase assays: NIH 3T3 cells were transfected using calcium phosphate coprecipitation with a wild-type or mutant bim promoter firefly-luciferase reporter plasmid and a Renilla luciferase transfection control. Cells were treated with low-dose insulin (0.2 μg mL⁻¹) and 100 nM VO-OHpic. Promoter activity was measured 2 and 4 hours after treatment using a dual-luciferase reporter assay system. [2]

Male C57BL6 mice (2-3 months old, weighing 21-25 g) were used. VO-OHpic was administered by intraperitoneal injection at a dosage of 10 μg/kg once, 30 minutes before ischemia. For the in vivo ischemia and reperfusion model, mice were anesthetized with pentobarbital (70 mg/kg, intraperitoneal). The left coronary artery was occluded for 30 minutes, followed by 120 minutes of reperfusion. Saline was used as control. At the end of the experiment, hearts were isolated for infarct size measurement. [3]
For measurement of left ventricular pressure, a Mikro-tip catheter was inserted into the left ventricle in anesthetized mice. Left ventricular systolic pressure, end-diastolic pressure, heart rate, positive maximal left ventricular pressure derivative (+dp/dt_m), and negative maximal left ventricular pressure derivative (-dp/dt_m) were recorded using a Powerlab Data Acquisition System and Chart 5 software. [3]
For immunoblotting, cardiac tissues were homogenized in lysis buffer (20 mM Tris pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM PMSF, 1 mM Na3VO4, 1% Triton). Proteins were detected using primary antibodies against p-Akt (S-473) and Akt, followed by horseradish peroxidase-conjugated secondary antibody and enhanced chemiluminescence. [3]
Myocardial infarct size was measured by incubating heart sections in 1.5% triphenyltetrazolium chloride (TTC) for 15 minutes at 37°C. Infarcted myocardium (white), area at risk (red), and area at non-risk (blue) were measured by computerized planimetry. Infarct size was calculated as (total weight of white tissue) / (total weight of white tissue + red tissue) × 100%. [3]

It was not possible to test concentrations of VO-OHpic above the IC50 for PTP-1B (which is in the micromolar range) due to cytotoxic effects observed at concentrations at or higher than 100 μM. VO-OHpic was noted to accelerate wound healing in fibroblasts, indicating it has the potential to alter PTEN functions related to cell migration and invasive properties, which is linked to its tumor suppressor function. [2]

[1]. Characterisation of the PTEN inhibitor VO-OHpic. J Chem Biol. 2010 Oct;3(4):157-63.

[2]. A small molecule inhibitor for phosphatase and tensin homologue deleted on chromosome 10 (PTEN). ACS Chem Biol. 2006 Dec 15;1(12):780-90.

[3]. PTEN inhibitors cause a negative inotropic and chronotropic effect in mice. Eur J Pharmacol. 2011 Jan 10;650(1):298-302.

VO-OHpic (vanadyl complex with two 3-hydroxypicolinic acid ligands and an oxo ligand) is a sterically demanding molecule. Its noncompetitive mode of inhibition distinguishes PTEN from other protein tyrosine phosphatases (PTPs), which are typically competitively inhibited by vanadium complexes. The similar Kic and Kiu values (27 nM vs 45 nM) indicate that the inhibitor binds with similar affinity to free enzyme and the enzyme-substrate complex. Unlike simple vanadyl salts (VOSO₄) that act as nonselective PTP inhibitors via vanadate delivery, VO-OHpic is the active compound itself, as shown by its high selectivity for PTEN and different inhibition mode. VO-OHpic has been employed to probe PTEN's role in PI3K-dependent signalling and PTEN-induced senescence. It has potential as a tool compound for studying PTEN as a drug target for diabetes, wound healing, asthma, neuroprotection, and certain cancers. [1]

2[C6H5NO3].HO2V.3[H2O]

416.21168

414.011

C, 34.80; H, 3.65; N, 6.76; O, 42.49; V, 12.30

476310-60-8

VO-OHPic;675848-25-6

66577002

Light green to green solid powder

9

13

0

26

384

0

C1=CC(=C(N=C1)C(=O)O)O.C1=CC(=C(N=C1)C(=O)O)O.[O-][V]=O

HCJXRYXYWPOIGP-UHFFFAOYSA-K

InChI=1S/2C6H5NO3.4H2O.O.V/c2*8-4-2-1-3-7-5(4)6(9)10;;;;;;/h2*1-3,8H,(H,9,10);4*1H2;;/q;;;;;;;+3/p-3

Hydroxy(oxo)vanadium 3-hydroxypyridine-2-carboxylic acid complex trihydrate

VO OHpic; VO-OHpic; VOOHpic

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)

DMSO: ~72 mg/mL (~173.4 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.02 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 (6.02 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% DMSO+40% PEG 300+2% Tween 80+ddH2O: 14mg/mL

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