PFKFB3 (IC50 = 137 nM)

PFK-158 (10 µM ; 24 hours; OV2008 and C13 cells) plus carboplatin (CBPt; 77-453 μM) significantly increases apoptosis in C13 (45%) and OV2008 cells (24.6%)[1].
PFK-158 (0-10 µM ; 24 hours; C13 and HeyA8MDR cells) treatment causes levels of lipid droplets (LD), p-PFKFB3, and p-cPLA2 to decrease in a dose-dependent manner[1].
PFK-158 (10 μM; 24 hours) has combined with Cisplatin to produce synergistic anti-proliferative effects in vitro in C13 and HeyA8MDR cells as opposed to OV2008 and HeyA8, respectively[1].
PFK-158 (0‐10 μM; 24 h) treatment induces autophagy induction in both C13 and HeyA8MDR cells as evidenced by the dose-dependent downregulation of p62/SQSTM1 and upregulation of LC3BII. The number of LDs is also decreased by PFK-158 treatment[1].

In rats and dogs, PFK158 is well tolerated, yielding a satisfactory pre-clinical therapeutic index. In numerous preclinical mouse models of tumors derived from humans as well as syngeneic murine models, PFK158 exhibits remarkable efficacy. The start of a phase I trial, which is currently in progress, was encouraged by IND-enabling safety and toxicity studies, which showed that PFK158 is well tolerated in rats and dogs.

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PFK158 alone suppresses tumor growth and ascites in vivo [1]
The efficacy of PFK158 alone and in combination with CBPt was evaluated on primary tumor growth and metastasis in HeyA8MDR‐bearing nude mice i.p. A marked reduction of tumor growth was observed in the combination treatment (Fig. 6 a‐b). At necropsy, tumor weight (Fig. 6 c), ascites volume (Fig. 6 d), and Ki67 staining (Fig. 6 e) showed that the combination treatment was more effective in reducing cancer progression compared to all other treatment groups. No significant body weight loss was observed in the control or any of the drug‐treatment groups (data not shown). Consistent with these data, the western blot analysis revealed decreased expression of p‐PFKFB3 and increased levels of the apoptotic marker cleaved‐caspase 3 in the combination treatment group (Fig. 6 f). Representative images of Bodipy (Fig. 6 g) and TUNEL staining(Fig. 6 h) from frozen sections of xenografts from the untreated controls as well as treatment groups showed that PFK158 treatment was more effective in reducing LDs while CBPt had little effect in inhibiting LDs. Similarly, there was more cell death in the combination group as assessed by TUNEL staining. These data demonstrate that PFK158 plus CBPt treatments lead to significantly enhanced antitumor activity in a gynecologic cancer mouse model.

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In vivo evaluation of synergy. [2]
First, we investigated the maximum tolerated doses (MTDs) of PFK-158 and its analogs or combination therapies in mice (Table S1). The results showed that the MTDs of 3PO, PFK-015, and PFK-158 were greater than or equal to 75 mg/kg, 30 mg/kg, and 60 mg/kg, respectively, when ICR mice received an intravenous (i.v.) injection. Additionally, the body weight gain of mice was not influenced by the compounds under MTDs or combination therapies (Fig. S2). Thus, a dose of 15 mg/kg (i.v.) was chosen as a safe dose for in vivo study. Mouse survival rates were monitored until day 3 postinfection. Compared with its analogs, PFK-158 showed a significant synergistic effect on colistin in systemically infected mice (Fig. 4C and F). In the K. pneumoniae H04 infection mouse model, colistin combined with PFK-158 treatment improved the survival rate from 10% (for 15 mg/kg PFK-158 or 1.3 mg/kg colistin monotherapy) to 40% (for the combination of 15 mg/kg PFK-158 and 1.3 mg/kg). The survival rate could also be improved from 10% to 30% when treatment with colistin combined with 3PO was administered (Fig. 4A). However, colistin combined with PFK-015 showed no influence on the survival rate in this model (Fig. 4B). In the HLCR E. cloacae D01 infection mouse model, the survival rate was significantly increased from 0% to 60% when colistin was combined with 15 mg/kg PFK-158 treatment. Colistin combined with 3PO or PFK-015 slightly improved the animal survival rates to 10% in the E. cloacae D01 infection model (Fig. 4C and D).

It was investigated whether PFK158 treatment could modify lipid pathways because increased glucose utilization in cancer promotes lipogenesis at several levels13 and because it might be another factor contributing to chemoresistance. Findings revealed that the PFK158 treatment dramatically decreased the number of LDs in the C13 and HeyA8MDR cells (Figs. 4c and d), which had more LDs than the chemosensitive cells (Fig. 4a and b). Interestingly, LDs were decreased in C13 and HeyA8MDR cells when PFKFB3 was genetically downregulated (Fig. 4e and g). According to the data, PFK158 and cisplatin together have synergistic antiproliferative effects in vitro in C13 and HeyA8MDR cells as compared to OV2008 and HeyA8, respectively (Fig. S3A-C and E-G, Supporting Information). In vitro and in vivo, PFK158 treatment causes lipophagy and makes chemoresistant cells more susceptible to the cytotoxic effects of chemotherapy. Notably, the results also demonstrated that more resistant cells than sensitive ones exhibit PFK158-induced chemosensitivity to carboplatin, which can be reversed by blocking autophagy with BafA. To sum up, this is one of the first investigations demonstrating that PFK158, a particular inhibitor of PFKFB3, simultaneously targets the lipogenic and glycolytic pathways—two highly active pathways in cancer—and encourages lipophagy to impede tumor growth.

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2‐[1‐14C]‐Deoxy‐d‐glucose uptake assay [1]
Cells seeded overnight were treated with increasing concentration of PFK158 (0–15 μM) for 1 h, washed with PBS and placed in glucose‐free media for 30 min. 2‐[1‐14C]‐deoxy‐d‐glucose (0.05 μCi/mL) was added and cells were further incubated for 1 h. After washing with PBS, cell lysates were prepared in 500 μL 0.5% SDS and 400 μL lysate was added to 5 mL scintillation fluid and counts were measured on a Tri‐Carb 2,910 liquid scintillation analyzer. Counts were then normalized to protein concentration measured by Bradford assay and data are represented as percent control of untreated cells.

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Antimicrobial agents and susceptibility test. [1]
All antibiotics, including colistin, polymyxin B, tigecycline, ceftazidime, cefepime, aztreonam, meropenem, amikacin, levofloxacin, and nitrofurantoin, were purchased commercially. Solvents and diluents for the preparation of antibiotics complied with Clinical and Laboratory Standards Institute (CLSI) guidelines. 3PO, PFK-015, and PFK158 were purchased commercially. The MICs of representative antibiotics were determined by the broth microdilution method in accordance with CLSI guidelines. The final inoculum in each well was approximately 5 × 105 CFU/ml. The microtiter plates were incubated at 37°C for 18 h, and the results were observed by the naked eye. The experiments were performed in triplicate on different days.

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Checkerboard analysis of combination effects. [1]
A clinical compound library (n = 688) was screened to identify the potentiators of colistin by a broth microdilution checkerboard assay. We examined the antibacterial activities of these combination therapies against both mcr-1-positive and mcr-1-negative colistin-resistant Enterobacteriaceae. Polymyxin-based combinations were prepared using 96-well round-bottom microtiter plates with drug concentrations that were 2-fold serially diluted. The two drugs were mixed in a 96-well plate, followed by the addition of a standard bacterial suspension at a final concentration of 5 × 105 CFU/ml in cation-adjusted Mueller-Hinton (CAMH) broth. After incubation for 18 h at 37°C, the results were also observed by the naked eye. The combination effects of polymyxins (PMB or CST) with PFK158 or its analogs were determined by calculating the fractional inhibitory concentration index (FICI) using the concentration combinations with the highest combination effects: FICI = (MIC of drug A in the combination/MIC of drug A alone) + (MIC of drug B in the combination/MIC of drug B alone). The antimicrobial combination was defined as synergistic when the FICI was ≤0.5; indifferent when 0.5 < FICI < 4, and antagonistic when the FICI was ≥4. The experiments were performed in triplicate on different days.

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Time-kill assays. [1]
Time-kill curve assays were performed with E. coli 13-43, K. pneumoniae H04, and E. cloacae D01, according to a method described previously by Lu et al. with minor modifications. Briefly, a culture of each isolate grown overnight was diluted with 3 ml CAMH broth to a final concentration of ∼106 CFU/ml. Next, colistin (at 2 μg/ml) and PFK158 (at the lowest concentrations that can show synergistic effects when combined with colistin), singly or in combinations, were added. Viable cell counts were determined 0, 1, 3, 5, 7, 9, and 24 h after incubation at 37°C by plating 10-μl serially diluted samples onto LB agar plates in triplicate. The results were recorded as log10 CFU per milliliter. The combination of colistin and PFK158 was considered synergistic if bacterial killing was 2 log10 units higher than the most active monotherapy.

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Scanning electron microscopy and transmission electron microscopy. [1]
SEM and TEM were employed to examine the effect of the colistin–PFK158 combination on the cellular morphology of the HLCR isolate E. cloacae D01. A log-phase culture was treated with 2 μg/ml colistin, 4 μg/ml PFK158, or both for 4 h in CAMH broth as described above for the time-kill studies. For the SEM and TEM studies, samples were transferred to 15-ml polypropylene tubes and centrifuged at 6,000 × g for 3 min. Supernatants were discarded, and bacterial pellets were resuspended and washed in 1 ml 2.5% glutaraldehyde in phosphate-buffered saline (PBS). The tubes were fixed overnight at 4°C. Once fixed, the tubes were centrifuged again at 6,000 × g for 3 min, the fixatives were removed, and bacterial pellets were finally resuspended in 1 ml PBS. SEM was conducted by using a scanning electron microscope, and TEM was conducted by using a transmission electron microscope.

Cell Line: OV2008 and C13 cells
Concentration: 10 µM
Incubation Time: 24 hours
Result: Combined with Carboplatin (CBPt) treatment resulted in significant increase in apoptosis.

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Cell viability and drug combination assay [1]
Cells were seeded in 96‐well plates, treated with increasing concentrations of drugs for 48 h and inhibitory concentration 50% (IC50) values were determined by MTT assays as previously described.19 To determine if PFK158 acts synergistically with cisplatin and carboplatin, constant ratio synergy studies were performed using the Chou–Talalay method20 and the combination index (CI) and dose reduction index (DRI) were calculated using CalcuSyn software.To determine the role of autophagy in PFK-158‐mediated sensitization of chemoresistant cells, cells were pretreated with 50 nM Bafilomycin A (BafA) for 2 h and were further treated with increasing concentrations of Carboplatin (CBPt) plus PFK-158. After 24 h, cell viability was assessed by MTT.

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Apoptosis assay [1]
Briefly, cells (1 × 106) were treated with PFK158, CBPt, Paclitaxel (PTX) alone and in combination, and Phosphatidyl‐serine externalization was analyzed by double staining the cells with FITC‐Annexin V and PI (5 μg/mL). Cells were acquired and analyzed by CellQuest Pro software as previously described.

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Cell imaging using 2‐NBDG [1]
Glucose uptake of the live cells was measured using 2‐NBDG as previously described.22 Briefly, cells were treated with 5 μMP PFK158 in glucose‐free medium for 30 min along with 2‐NBDG and then examined under Zeiss‐LSM 510 fluorescence microscope. The fluorescent intensities were calculated using Image J software as previously reported.

Female athymic nude mice (nu/nu) (5-6 weeks old) injected with HeyA8MDR cells[1]
15 mg/kg
Intraperitoneal injection; once a week; for 4 weeks

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Female athymic nude mice (nu/nu) (5–6 weeks old) were randomized in six groups (n = 10) and HeyA8MDR cells (3 × 106) were injected intraperitoneally (i.p). Seven days after i.p., mice were treated with (i) PBS for control group, (ii) 25 mg/kg of PFK158 every 3rd day, (iii) 51 mg/kg of CBPt every 3rd day, (iv) PTX (15 mg/kg) every 5th day, (v) combination of CBPt (51 mg/kg) and PFK-158 (15 mg/kg) once a week, and (vi) PTX (5 mg/kg) and PFK-158 (25 mg/kg) once a week. The treatments were continued until the end of the study (28 days), however, control mice were sacrificed during week 3 as the tumor burden exceeded 10% of their body weight. Tumors and tissues were excised and preserved either in formalin or −80 °C. [1]

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All compounds were prepared in 5% ethanol, 5% Cremophor, and 90% D5W (5% dextrose in water). The safety study was conducted with female ICR mice. Mice were intravenously injected with a solvent, 75 mg/kg 3PO, 30 mg/kg PFK-015, or 60 mg/kg PFK158 (n = 8 per group). The body weight changes of the mice were monitored for a week postinjection. Female ICR mice (body weights of 18 to 20 g) were also used for the mouse systemic infection model. Mice were infected intraperitoneally with a 0.5-ml K. pneumoniae H04 or E. cloacae D01 bacterial suspension (100% minimum lethal dose) in 5% mucin. After 1 h of infection, colistin (1 mg/kg for K. pneumoniae H04 infection and 1.3 mg/kg for E. cloacae D01 infection), 3PO, PFK-015, or PFK158 (15 mg/kg), singly or in combination, was injected intravenously into the mice (n = 10 per group). A group of mice was treated with the same solvent as a control group. The experiments were performed in triplicate on different days. A log rank test was applied to compare the survival distributions of different samples.[2]

[1]. Therapeutic targeting of PFKFB3 with a novel glycolytic inhibitor PFK158 promotes lipophagy and chemosensitivity in gynecologic cancers. Int J Cancer. 2019 Jan 1;144(1):178-189.

[2]. Synergistic Effect of Colistin Combined with PFK-158 against Colistin-Resistant Enterobacteriaceae. Antimicrob Agents Chemother. 2019 Jun 24;63(7). pii: e00271-19.

[3]. Pfkfb3 inhibitor and methods of use as an anti-cancer therapeutic. WO2013148228A1.

PFK-158 is a small molecule drug with a maximum clinical trial phase of I and has 1 investigational indication.

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Metabolic alterations are increasingly recognized as important novel anti-cancer targets. Among several regulators of metabolic alterations, fructose 2,6 bisphosphate (F2,6BP) is a critical glycolytic regulator. Inhibition of the active form of PFKFB3ser461 using a novel inhibitor, PFK158 resulted in reduced glucose uptake, ATP production, lactate release as well as induction of apoptosis in gynecologic cancer cells. Moreover, we found that PFK158 synergizes with carboplatin (CBPt) and paclitaxel (PTX) in the chemoresistant cell lines, C13 and HeyA8MDR but not in their chemosensitive counterparts, OV2008 and HeyA8, respectively. We determined that PFK158-induced autophagic flux leads to lipophagy resulting in the downregulation of cPLA2, a lipid droplet (LD) associated protein. Immunofluorescence and co-immunoprecipitation revealed colocalization of p62/SQSTM1 with cPLA2 in HeyA8MDR cells uncovering a novel pathway for the breakdown of LDs promoted by PFK158. Interestingly, treating the cells with the autophagic inhibitor bafilomycin A reversed the PFK158-mediated synergy and lipophagy in chemoresistant cells. Finally, in a highly metastatic PTX-resistant in vivo ovarian mouse model, a combination of PFK158 with CBPt significantly reduced tumor weight and ascites and reduced LDs in tumor tissue as seen by immunofluorescence and transmission electron microscopy compared to untreated mice. Since the majority of cancer patients will eventually recur and develop chemoresistance, our results suggest that PFK158 in combination with standard chemotherapy may have a direct clinical role in the treatment of recurrent cancer.[1]

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In conclusion, this is one of the first studies to show that PFK158, a specific inhibitor of PFKFB3, simultaneously targets both the glycolytic and lipogenic pathways, two pathways that are very active in cancer, and promotes lipophagy to inhibit tumor growth. Since resistance to chemotherapy is a major impediment in prolonging survival cancer patients, it is imperative to identify therapeutic strategies to overcome chemoresistance. Toward this aim, the synergy that we observed between PFK158 and carboplatin in chemoresistant cells has tremendous clinical relevance as it could represent a novel therapeutic option for patients with recurrent chemoresistant disease.[1]

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As increasing numbers of colistin-resistant bacteria emerge, new therapies are urgently needed to treat infections caused by these pathogens. The discovery of new combination therapies is one important way to solve such problems. Here, we report that the antitumor drug PFK-158 and its analogs PFK-015 and 3PO can exert synergistic effects with colistin against colistin-resistant Enterobacteriaceae, including mcr-1-positive or high-level-colistin-resistant (HLCR) isolates, as shown by a checkerboard assay. The results of a time-kill assay revealed that colistin combined with PFK-158 continuously eliminated colistin-resistant Escherichia coli 13-43, Klebsiella pneumoniae H04, and Enterobacter cloacae D01 in 24 h. Images from scanning electron microscopy (SEM) at 5 h postinoculation confirmed the killing effect of the combination. Finally, in vivo treatment showed that PFK-158 had a better synergistic effect than its analogs. Compared to the corresponding rates after colistin monotherapy, the survival rates of systemically infected mice were significantly increased 30% or 60% when the mice received an intravenous injection of colistin in combination with 15 mg/kg of body weight PFK-158. These results have important implications for repurposing PFK-158 to combat colistin resistance.[2]

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In conclusion, one new class of potentiators of colistin was evaluated both in vitro and in vivo. PFK-158 not only decreased the MICs of colistin to eradicate both colistin-susceptible and colistin-resistant (mcr-1 or HLCR) Enterobacteriaceae in vitro but also enhanced the efficacy of colistin in vivo. This finding might shed light on the discovery of combination therapies for infections caused by colistin-resistant pathogens.[2]

C18H11F3N2O

328.08

328.082

C, 65.85; H, 3.38; F, 17.36; N, 8.53; O, 4.87

1462249-75-7

71730058

White to light yellow solid powder

1.3±0.1 g/cm3

466.3±45.0 °C at 760 mmHg

235.8±28.7 °C

0.0±1.2 mmHg at 25°C

1.624

3.55

0

6

3

24

471

0

FC(C1C([H])=C([H])C2C([H])=C([H])C(/C(/[H])=C(\[H])/C(C3C([H])=C([H])N=C([H])C=3[H])=O)=NC=2C=1[H])(F)F

IAJOMYABKVAZCN-AATRIKPKSA-N

InChI=1S/C18H11F3N2O/c19-18(20,21)14-3-1-12-2-4-15(23-16(12)11-14)5-6-17(24)13-7-9-22-10-8-13/h1-11H/b6-5+

(E)-1-pyridin-4-yl-3-[7-(trifluoromethyl)quinolin-2-yl]prop-2-en-1-one

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 mg/mL (6.09 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.0 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 mg/mL (6.09 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.0 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 mg/mL (6.09 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 20.0 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.)