STAT5b（IC50 = 82 nM)

The affinity of stafib-2 for STAT5b is remarkably high (Ki=8.8 nM)[1]. In K562 and MDA-MB-231 cells, stafib-2 (3–10 μM; 4-48 h) does not exhibit any discernible activity[1].
The binding site of Stafib-2 was validated using combined isothermal titration calorimetry (ITC) and protein point mutant analysis, representing the first time that functional comparison of wild-type versus mutant protein by ITC has been used to characterize the binding site of a small-molecule ligand of a STAT protein with amino acid resolution. The prodrug Pomstafib-2 selectively inhibits tyrosine phosphorylation of STAT5b in human leukaemia cells and induces apoptosis in a STAT5-dependent manner. We propose Pomstafib-2, which currently represents the most active, selective inhibitor of STAT5b activation available, as a chemical tool for addressing the fundamental question of which roles the different STAT5 proteins play in various cell processes.[1]

Fluorescence polarization (FP) assays [1]
The ability of the test compounds to displace fluorophore-labelled peptides (final concentration: 10 nM) from their respective binding proteins was analysed as previously described. Peptide sequences were: STAT1: 5-carboxyfluorescein-GY(PO3H2)DKPHVL; STAT3: 5-carboxyfluorescein-GY(PO3H2)LPQTV-NH2; STAT4: 5-carboxyfluorescein-GY(PO3H2)LPQNID-OH; STAT5a and STAT5b: 5-carboxyfluorescein-GY(PO3H2)LVLDKW; STAT6: 5-carboxyfluorescein-GY(PO3H2)VPWQDLI-OH; Lck SH2: 5-carboxyfluorescein-GY(PO3H2)EEIP. STAT2 was not analysed due to protein instability. Final protein concentrations: STAT1: 420 nM; STAT3: 270 nM; STAT4: 130 nM; STAT5a: 130 nM; STAT5b: 100 nM; STAT6: 310 nM; Lck SH2: 30 nM. These concentrations correspond to the Kd-values of the respective protein-peptide interactions. Pipetting was carried out in part using a Biomek FX robot. Proteins and compounds were incubated for 1 h before addition of the fluorescent-labelled peptides. After an additional hour, fluorescence polarization was measured using an Infinite F500 plate reader. Final buffer concentrations: 10 mM Tris (pH 8.0), 50 mM NaCl, 1 mM EDTA, 1 mM DTT, 0.1% Nonidet P-40 substitute, 2% DMSO. Changes in FP were converted to percent inhibition based on peptide-protein binding curve fits. Ki-values were calculated from IC50 data using the published equation.

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Isothermal titration calorimetry (ITC) [1]
ITC experiments were run on a MicroCal VP-ITC calorimeter. Typical titrations setting were: 25 °C cell temperature, 150 s initial delay, ca. 20 µM STAT protein in 10 mM Tris, 50 mM NaCl, pH 8.0, 200 µM of compound 4 as a tetra sodium salt, stirring speed 300 rpm, reference power to 20 µcal/s. All solutions were degassed before the experiments. The resulting data were analysed by NITPIC and SEDPHAT34 and fitted with the one-site binding model, whilst defining the concentration of 4 as fixed. A low-noise thermogram integration approach was used. Illustrations were made using GUSSI.

Western Blots [1]
Transfection of cultured K562 cells and Western blotting was performed as previously described. K562 cells were transfected with plasmid encoding either STAT5a-GFP or STAT5b-GFP, using Fugene HD Transfection Reagent (1 x 106 cells per well in 1 ml medium with a 4:1 ratio of Fugene:DNA). 24 h later, the cells were treated with compound or DMSO for 4 h (final DMSO concentration: 0.2%). After harvesting, cells were lysed (lysis buffer composition: 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10 mM Na4P2O7, 10% glycerol, 1% Triton X-100, 1 mM EDTA, 100 ng/mL aprotinin, 1 mM Na3VO4, 10 mM NaF, 1 mM PMSF). The cell lysate components were separated by SDS-PAGE (10%) and then transferred to a nitrocellulose membrane. Primary antibodies (phospho-STAT5; STAT5; β-Actin) were detected using α-rabbit-HRP secondary antibody and ECL, and visualized using an ImageQuant imager. ImageJ software (NIH) was used for signal quantitation.

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Apoptosis assay [1]
Apoptosis assays were performed as previously described16. In brief, K562 cells (2.5 × 105 cells per well) or MDA-MB-231 cells (1 × 105 cells per well) were seeded in 24-well tissue culture plates, and treated with compound 8 at the indicated concentrations (final DMSO concentration: 0.2%) for 48 h. Cells were harvested after 48 h. MDA-MB-231 cells were washed twice with warm phosphate buffered saline (PBS), followed by incubation with Accutase at 37 °C for 10 min. Neutralization of Accutase and cell resuspension was carried out with the cell culture supernatant from each well. After harvesting, cells were centrifuged at 3000 rpm at 4 °C for 5 min, washed twice with cold PBS, and centrifuged again. Cells were stained using the PE Annexin V Apoptosis Detection Kit I. Cells were resuspended in binding buffer and incubated with PE Annexin V and 7-AAD at 4 °C for 30 min. Apoptosis was measured using a LSR II flow cytometer.

[1]. Rational development of Stafib-2: a selective, nanomolar inhibitor of the transcription factor STAT5b. Sci Rep. 2017 Apr 11;7(1):819.

The transcription factor STAT5b is a target for tumour therapy. We recently reported catechol bisphosphate and derivatives such as Stafib-1 as the first selective inhibitors of the STAT5b SH2 domain. Here, we demonstrate STAT5b binding of catechol bisphosphate by solid-state nuclear magnetic resonance, and report on rational optimization of Stafib-1 (Ki = 44 nM) to Stafib-2 (Ki = 9 nM). The binding site of Stafib-2 was validated using combined isothermal titration calorimetry (ITC) and protein point mutant analysis, representing the first time that functional comparison of wild-type versus mutant protein by ITC has been used to characterize the binding site of a small-molecule ligand of a STAT protein with amino acid resolution. The prodrug Pomstafib-2 selectively inhibits tyrosine phosphorylation of STAT5b in human leukaemia cells and induces apoptosis in a STAT5-dependent manner. We propose Pomstafib-2, which currently represents the most active, selective inhibitor of STAT5b activation available, as a chemical tool for addressing the fundamental question of which roles the different STAT5 proteins play in various cell processes.[1]

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We report the rational optimization of the STAT5b SH2 domain inhibitor Stafib-1 (2) to Stafib-2 (4), which displays significantly increased activity whilst maintaining high selectivity over the closely related SH2 domain of STAT5a. We provide the first application of solid-state NMR for the analysis of ligand binding to an SH2 domain. The high affinity of Stafib-2 for STAT5b allowed us to perform the first comparative analysis of binding between a small-molecule STAT SH2 domain ligand and wild-type versus mutant STAT proteins by ITC, providing experimental validation of the hydrophilic and hydrophobic Stafib-2 binding pockets of STAT5b. The pivaloyloxymethylester Pomstafib-2 (8) inhibits STAT5b phosphorylation in cultured human leukaemia cells with an IC50 of only 1.5 µM, without significantly affecting the phosphorylation of STAT5a, and increases the apoptotic rate of human leukaemia cells in a STAT5-dependent manner. Since Pomstafib-2 (8) currently represents the most active, selective inhibitor of STAT5b activation, we propose its use as a chemical tool to dissect the overlapping and non-redundant functions of the two STAT5 proteins in living cells.[1]

C28H26N2O12P2

644.4597697258

644.096

C, 52.18; H, 4.07; N, 4.35; O, 29.79; P, 9.61

2097938-74-2

129896883

White to off-white solid powder

1.8

6

12

13

44

1020

0

P(=O)(O)(O)OC1=C(C=CC(=C1)CNC(COC1C=CC(C(NC2C=CC(=CC=2)OC2C=CC=CC=2)=O)=CC=1)=O)OP(=O)(O)O

LJGIDZSIOSYQMR-UHFFFAOYSA-N

InChI=1S/C28H26N2O12P2/c31-27(29-17-19-6-15-25(41-43(33,34)35)26(16-19)42-44(36,37)38)18-39-22-11-7-20(8-12-22)28(32)30-21-9-13-24(14-10-21)40-23-4-2-1-3-5-23/h1-16H,17-18H2,(H,29,31)(H,30,32)(H2,33,34,35)(H2,36,37,38)

[4-[[[2-[4-[(4-phenoxyphenyl)carbamoyl]phenoxy]acetyl]amino]methyl]-2-phosphonooxyphenyl] dihydrogen phosphate

Stafib-2; 2097938-74-2; Benzamide, 4-[2-[[[3,4-bis(phosphonooxy)phenyl]methyl]amino]-2-oxoethoxy]-N-(4-phenoxyphenyl)-; 4-((2-(4-((4-Phenoxyphenyl)carbamoyl)phenoxy)acetamido)methyl)-1,2-phenylene bis(dihydrogen phosphate); [4-[[[2-[4-[(4-phenoxyphenyl)carbamoyl]phenoxy]acetyl]amino]methyl]-2-phosphonooxyphenyl] dihydrogen phosphate; Stafib-2?; DTXSID601118678

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: 50 mg/mL (77.58 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (3.88 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 (3.88 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 (3.88 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.)