PFKFB3 (IC50 = 155 nM)

3PO does not inhibit the activity of purified PFK-1; instead, it inhibits the PFKFB3 isozyme mainly through competition with Fru-6-P. When compared to normal human bronchial epithelial cells, 3PO selectively cytostatically affects ras-transformed human bronchial epithelial cells, thereby significantly attenuating the proliferation of several human malignant hematopoietic and adenocarcinoma cell lines (IC50, 1.4-24 μmol/L). 3PO can result in phase arrest in G2-M[1].

When tumor-bearing mice are given 3PO intraperitoneally (0.07 mg/g), the intracellular concentration of Fru-2,6-BP, the uptake of glucose, and the growth of established tumors are all significantly reduced in vivo. It inhibits leukemia, lung adenocarcinoma, and breast adenocarcinoma cells' ability to grow tumorigenic in vivo[1]. After intravenously administering 3PO to C57Bl/6 mice, the following PK properties are investigated: clearance CL=2312 mL/min/kg, T_1/2=0.3 hr, C_max=113 ng/ml, and AUC_0-inf=36 ng/hr/ml. According to reports, 3PO exhibits strong activity against a leukemia mouse model that is highly relevant[2].

PFKFB3 Enzymatic Assays[1]
PFKFB3 protein activity was measured by an enzyme-coupled kinetics assay incorporating pyruvate kinase and lactate dehydrogenase as described previously. Control reactions for 3PO inhibition contained increasing amounts of 3PO without addition of PFKFB3. The enzyme kinetics module for SigmaPlot 9.0 was used to calculate the kinetic variables for PFKFB3 and 3PO inhibition (Vmax, Km, and Ki). The data represented are the mean ± SD from triplicate measurements from two independent experiments.

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Generation of FLAG-PFKFB3 Construct for Mammalian Expression[1]
FLAG-PFKFB3 containing the complete PFKFB3 coding sequence and FLAG epitope at its NH2 terminus was subcloned into the BamHI/HindIII restriction sites within the retroviral Tet response vector pRevTRE. Recombinant retrovirus was produced by Lipofectamine-mediated transfection of the pRevTRE-FLAG-PFKFB3 construct into PT67 packaging cell lines. To create Jurkat cell lines that have stably integrated and express inducible FLAG-PFKFB3, the cells were infected with recombinant retrovirus containing FLAG-PFKFB3, and stable clones were selected in the presence of 400 μg/mL hygromycin.

In RPMI 1640 supplemented with 10% fetal bovine serum and 50 μg/mL gentamicin sulfate, Jurkat cells are plated at a density of 1 × 10⁵/mL. The cells are treated with either vehicle or 10 μmol/L 3PO for a duration of 0, 4, 8, 16, 24, or 36 hours. The cell cycle is analyzed.

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In vitro 3PO Growth Inhibition [1]
All cell lines were plated at 1 × 105/mL in the appropriate medium. For suspension cells, 3PO was added immediately to the medium, whereas 3PO treatment was initiated the following day for adherent cell lines. For dose-dependent experiments, 3PO was added in increasing concentrations for 36 h. For time-dependent experiments, 10 μmol/L 3PO was added at time 0, 4, 8, 16, 24, or 36 h. For PFKFB3 overexpression studies, Jurkat cells containing the FLAG-PFKFB3 expression vector or a control plasmid were induced by addition of doxycycline (1 μg/mL) 24 h before 3PO incubation. Cells were then collected 48 h after treatment, and cell number and viability were determined by trypan blue exclusion. IC50s were calculated as the 3PO concentration needed for 50% of vehicle-treated cell growth. The data represented are the mean ± SD from triplicate measurements from three independent experiments.

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Cell Cycle Analysis and Flow Cytometry [1]
Jurkat cells were plated at 1 × 105/mL in RPMI 1640 supplemented with 10% fetal bovine serum and 50 μg/mL gentamicin sulfate. Cells were immediately treated with vehicle or 10 μmol/L 3PO for 0, 4, 8, 16, 24, or 36 h. Cell cycle analysis was done according to the manufacturer's protocol using Vybrant DyeCycle Orange stain.

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Fru-2,6-BP and Lactate Measurements [1]
Jurkat cells were plated at 1 × 105/mL and immediately incubated with 10 μmol/L 3PO for 0, 4, 8, 16, 24, or 36 h. Media samples were collected and lactate levels were measured using a lactate oxidase-based colorimetric assay read at 540 nm according to the manufacturer's instructions and normalized to protein concentration. Fru-2,6-BP assays were done as described previously.

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2-Deoxyglucose Uptake [1]
Jurkat cells were plated at 1 × 105/mL in RPMI 1640 supplemented with 10% fetal bovine serum and 50 μg/mL gentamicin sulfate. Cells were immediately treated with vehicle or 10 μmol/L 3PO for the indicated time periods and subsequently placed in glucose-free RPMI 1640 for 30 min. 14C-2-deoxyglucose (0.25 μCi/mL) was added for an additional 60 min and cells were then washed three with ice-cold RPMI 1640 containing no glucose. Cell lysates were collected in 500 μL of 0.1% SDS, and scintillation counts (counts/min) were measured on 400 μL of lysate. Counts were normalized to protein concentration, and data are represented as mean ± SD from duplicate measurements from two independent experiments.

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Whole-Cell ATP, NAD+, and NADH Determination [1]
Jurkat cells were plated at 1 × 105/mL and immediately incubated with 10 μmol/L 3PO for the indicated time periods. ATP levels were determined using the ATP determination kit according to the manufacture's protocol and NAD+ and NADH levels were measured using the EnzyChrom NAD+/NADH assay kit on 1 × 106 cells for both vehicle and 3PO-treated samples at all time points.

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Metabolite Extraction for Nuclear Magnetic Resonance [1]
Jurkat cells were treated with vehicle or 10 μmol/L 3PO in the presence of 13C-glucose for 36 h. The cells were counted and equal numbers of cells were pelleted, washed twice with cold PBS to remove adhering medium, and flash frozen in liquid N2. The cold pellet was extracted with 10% ice-cold trichloroacetic acid (twice) followed by lyophilization. Dry extract was redissolved in 0.35 mL D2O and loaded into a 5 mm Shigemi tube.

tumor bearing mice (BALB/c nude mice or C57Bl/6 female mice background)
0.07 mg/g
i.p.

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In vivo Studies [1]
Exponentially growing MDA-MB231 and HL-60 cells were collected, washed, and resuspended in PBS at 20 × 107/mL. Cells were then mixed 1:1 with Matrigel, and 0.1 mL of the cell suspension was injected s.c. (1 × 107 cells) into female BALB/c nude mice (20 g). Exponentially growing Lewis lung carcinoma cells were collected, washed twice, and resuspended in PBS (1 × 107/mL). C57Bl/6 female mice (20 g) were injected s.c. with 0.1 mL of the suspension. Body weight and tumor growth were monitored daily throughout the study. Tumor masses were determined by measurement with Vernier calipers using the formula: mass (mg) = [width2 (mm) × length (mm)] / 2. Mice with established tumors (between 130 and 190 mg) were randomized into vehicle control or 3PO-treated groups. Vehicle control groups received i.p. injections of 50 μL DMSO, whereas treated groups received 0.07 mg/g 3PO in 50 μL DMSO at the indicated time points. All tumor experiments were conducted three times and the data presented are from one experiment.

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In vivo Fru-2,6-BP Measurement[1]
C57Bl/6 female mice (20 g) were injected s.c. with 1 × 106 Lewis lung carcinoma cells. When xenografts were measured to have a mass of 150 to 180 mg, mice were randomized and given i.p. injections of vehicle DMSO or 0.07 mg/g 3PO. Four hours after injection, tumors were removed and homogenized in 1 volume of 0.05 mol/L NaOH and subsequently mixed with 1 volume of 0.1 mol/L NaOH. Fru-2,6-BP assays were done as described previously.

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Micro-Positron Emission Tomography[1]
Lewis lung carcinoma xenograft-bearing mice were given i.p. injections of 50 μL DMSO or 0.07 mg/g 3PO in DMSO. After 30 min, mice were injected i.p. with 18F-2-DG (150 μCi, 100 μL in H2O) and subsequently anesthetized after 15 min with 2% isoflurane in oxygen. The mice were then transferred to a R-4 Rodent Scanner micro-positron emission tomography (CTI Concorde Microsystems; n = 3).

[1]. Mol Cancer Ther . 2008 Jan;7(1):110-20.

[2]. Mol Cancer Ther . 2013 Aug;12(8):1461-70.

3PO is a member of the class of pyridines that is pyridine substituted by a 3-oxo-3-(pyridin-4-yl)prop-1-en-1-yl group at position 3. An inhibitor of PFKFB3 kinase, an enzyme with a key role in glycolysis. It has a role as an antineoplastic agent, an angiogenesis inhibitor, an autophagy inducer, an apoptosis inducer and an EC 2.7.1.105 (6-phosphofructo-2-kinase) inhibitor. It is a member of pyridines and an enone.

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6-phosphofructo-1-kinase, a rate-limiting enzyme of glycolysis, is activated in neoplastic cells by fructose-2,6-bisphosphate (Fru-2,6-BP), a product of four 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase isozymes (PFKFB1-4). The inducible PFKFB3 isozyme is constitutively expressed by neoplastic cells and required for the high glycolytic rate and anchorage-independent growth of ras-transformed cells. We report herein the computational identification of a small-molecule inhibitor of PFKFB3, 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO), which suppresses glycolytic flux and is cytostatic to neoplastic cells. 3PO inhibits recombinant PFKFB3 activity, suppresses glucose uptake, and decreases the intracellular concentration of Fru-2,6-BP, lactate, ATP, NAD+, and NADH. 3PO markedly attenuates the proliferation of several human malignant hematopoietic and adenocarcinoma cell lines (IC50, 1.4-24 micromol/L) and is selectively cytostatic to ras-transformed human bronchial epithelial cells relative to normal human bronchial epithelial cells. The PFKFB3 enzyme is an essential molecular target of 3PO because transformed cells are rendered resistant to 3PO by ectopic expression of PFKFB3 and sensitive to 3PO by heterozygotic genomic deletion of PFKFB3. Importantly, i.p. administration of 3PO (0.07 mg/g) to tumor-bearing mice markedly reduces the intracellular concentration of Fru-2,6-BP, glucose uptake, and growth of established tumors in vivo. Taken together, these data support the clinical development of 3PO and other PFKFB3 inhibitors as chemotherapeutic agents.[1]

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In human cancers, loss of PTEN, stabilization of hypoxia inducible factor-1α, and activation of Ras and AKT converge to increase the activity of a key regulator of glycolysis, 6-phosphofructo-2-kinase (PFKFB3). This enzyme synthesizes fructose 2,6-bisphosphate (F26BP), which is an activator of 6-phosphofructo-1-kinase, a key step of glycolysis. Previously, a weak competitive inhibitor of PFKFB3, 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO), was found to reduce the glucose metabolism and proliferation of cancer cells. We have synthesized 73 derivatives of 3PO and screened each compound for activity against recombinant PFKFB3. One small molecule, 1-(4-pyridinyl)-3-(2-quinolinyl)-2-propen-1-one (PFK15), was selected for further preclinical evaluation of its pharmacokinetic, antimetabolic, and antineoplastic properties in vitro and in vivo. We found that PFK15 causes a rapid induction of apoptosis in transformed cells, has adequate pharmacokinetic properties, suppresses the glucose uptake and growth of Lewis lung carcinomas in syngeneic mice, and yields antitumor effects in three human xenograft models of cancer in athymic mice that are comparable to U.S. Food and Drug Administration-approved chemotherapeutic agents. As a result of this study, a synthetic derivative and formulation of PFK15 has undergone investigational new drug (IND)-enabling toxicology and safety studies. A phase I clinical trial of its efficacy in advanced cancer patients will initiate in 2013 and we anticipate that this new class of antimetabolic agents will yield acceptable therapeutic indices and prove to be synergistic with agents that disrupt neoplastic signaling.[2]

C13H10N2O

210.2313

210.079

C, 74.27; H, 4.79; N, 13.33; O, 7.61

18550-98-6

18550-98-6; 13309-08-5

5720233

Light yellow to yellow solid powder

1.2±0.1 g/cm3

387.8±42.0 °C at 760 mmHg

191.3±34.3 °C

0.0±0.9 mmHg at 25°C

1.637

1.51

0

3

3

16

257

0

O=C(/C(/[H])=C(\[H])/C1=C([H])N=C([H])C([H])=C1[H])C1C([H])=C([H])N=C([H])C=1[H]

UOWGYMNWMDNSTL-ONEGZZNKSA-N

InChI=1S/C13H10N2O/c16-13(12-5-8-14-9-6-12)4-3-11-2-1-7-15-10-11/h1-10H/b4-3+

(E)-3-pyridin-3-yl-1-pyridin-4-ylprop-2-en-1-one

3PO; 3-PO; 18550-98-6; 13309-08-5; (E)-3PO; 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one; (E)-3-pyridin-3-yl-1-pyridin-4-ylprop-2-en-1-one; 2-Propen-1-one, 3-(3-pyridinyl)-1-(4-pyridinyl)-; CHEMBL3105848;

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: 4~60 mg/mL (199.8~285.4 mM)
Ethanol: ~11 mg/mL (52.3 mM)

Solubility in Formulation 1: ≥ 3 mg/mL (14.27 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 30.0 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: ≥ 3 mg/mL (14.27 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 30.0 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: ≥ 3 mg/mL (14.27 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 30.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 2% DMSO+40% PEG 300+2% Tween 80+ddH2O: 2mg/mL

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