KAN0438757 (72 h) reduces the cell viability of Miapaca-2, PANC1, SW620, U-266, and AMO-1 (IC50: 2.75, 3.83, 7.50, 5.08, 11.53 μM, respectively) [1]. KAN0438757 (10 μM, 6 KAN0438757 (50 μM, 12 hours) lowers homologous recombination (HR) activity and increases ionizing radiation (IR)-induced γH2AX foci levels in U2OS cells [1]. KAN0438757 (50 μM, 12 hours ) can reduce HCT-116, SW-1463 and HUVEC KAN0438757 (0-50 μM, 24 hours) can reduce HCT-116 carbohydrates and glycolysis in HUVEC [2].

KAN0438757 (72 h) reduces the cell viability of Miapaca-2, PANC1, SW620, U-266, and AMO-1 (IC50: 2.75, 3.83, 7.50, 5.08, 11.53 μM, respectively) [1]. KAN0438757 (10 μM, 6 KAN0438757 (50 μM, 12 hours) lowers homologous recombination (HR) activity and increases ionizing radiation (IR)-induced γH2AX foci levels in U2OS cells [1]. KAN0438757 (50 μM, 12 hours ) can reduce HCT-116, SW-1463 and HUVEC KAN0438757 (0-50 μM, 24 hours) can reduce HCT-116 carbohydrates and glycolysis in HUVEC [2].

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The prodrug KAN0438757 (designed for increased cell permeability and intracellular hydrolysis to the active acid KAN0438241) significantly suppresses intracellular fructose-2,6-bisphosphate (F-2,6-P2) levels with sub-μM IC50 values in pancreatic (MIA PaCa-2), gastric (NUGC-3), and colon (SW620) cancer cell lines, whereas the acid form KAN0438241 was only weakly active in cells [1].
KAN0438757 reduces the viability of several cancer cell lines (MIA PaCa-2, PANC-1, SW620, SW480) following prolonged (72 h) incubation, as measured by Sulforhodamine B assay [1].
Inhibition of PFKFB3 by KAN0438757 (10 μM) impairs the ionizing radiation (IR)-induced recruitment of homologous recombination (HR) repair factors RPA32 and RAD51 to nuclear foci in U2OS cells [1].
KAN0438757 treatment decreases HR repair activity to about 10% compared to vehicle-treated cells in the DR-GFP reporter assay in U2OS cells, an effect more potent than ATR inhibition [1].
Treatment with KAN0438757 prior to IR results in significantly higher levels of γH2AX foci (a marker of DNA double-strand breaks) at 24 h post-IR in U2OS cells, indicative of reduced DNA repair [1].
KAN0438757 induces radiosensitization in clonogenic survival assays. Pre-treatment of U2OS cells with KAN0438757 (10 μM) prior to IR leads to a dose-dependent reduction in colony formation [1].
The radiosensitizing effect of KAN0438757 is selective for transformed cells. In isogenic cell pairs, KAN0438757 treatment combined with IR significantly decreased clonogenic survival of transformed BJ RAS cells at concentrations that only marginally affected non-transformed BJ TERT (hTERT-immortalized) cells. In contrast, ATM or ATR inhibition combined with IR severely reduced survival of both transformed and non-transformed cells [1].
Overexpression of RAD51 rescued the clonogenic survival of BJ RAS cells treated with KAN0438757 at its IC50 concentration combined with IR, suggesting that the toxicity is linked to loss of HR function [1].
KAN0438757 treatment impairs IR-induced increase in EdU (thymidine analogue) incorporation in the G2/M phase of U2OS cells, suggesting a role in deoxynucleotide incorporation during repair synthesis [1].
KAN0438757 treatment abolishes the IR-induced co-localization between PFKFB3 and the RRM2 subunit of ribonucleotide reductase (RNR) in nuclear foci, and knockdown of PFKFB3 also blocks RRM2 recruitment, placing PFKFB3 activity upstream of RRM2 recruitment to damage sites [1].
Co-immunoprecipitation experiments show an interaction between FLAG-tagged PFKFB3 and RRM2 at 2 h post-IR, but not in non-IR conditions [1].
KAN0438757 treatment (10-30 μM, 4 h) decreases EdU incorporation intensity per cell in U2OS cells, similar to the effect of hydroxyurea (HU), indicating impaired DNA replication [1].
DNA fiber assays show that KAN0438757 treatment (10 μM, 24 h) severely decreases the speed of replication forks in U2OS cells. This impairment is restored by supplementation with nucleosides (adenosine, cytidine, guanosine, uridine) during treatment [1].
KAN0438757 treatment (10 μM) of U2OS cells for 4 h and 24 h results in a 50-75% decrease in the levels of all four deoxynucleotide triphosphates (dNTPs) [1].
In contrast to hydroxyurea (HU), KAN0438757 inhibition does not induce RPA-coated ssDNA, p53 induction, or phosphorylation of ATR, ATM, Chk1, or H2AX, suggesting it causes replication fork stalling without immediate collapse or checkpoint activation [1].
Cellular Thermal Shift Assay (CETSA) confirms intracellular target engagement of KAN0438757 for PFKFB3 in U2OS cells at concentrations consistent with its cellular activity, and this engagement persists at 24 h and 72 h of treatment [1].

In the C57BL6/N model, KAN0438757 (ip, 10; 25; 50 mg/kg) was well tolerated and did not exhibit any significant systemic toxic effects [2].

The kinase activity of recombinant human PFKFB3 was quantified using an ADP-Glo assay. The assay measures the production of ADP and F-2,6-P2 from ATP and fructose-6-phosphate (F6P). The reaction is terminated, and remaining ATP is depleted. ADP is then converted back to ATP, and the newly synthesized ATP is measured via a luciferase/luciferin reaction, with luminescence proportional to kinase activity. Inhibitors were pre-incubated with the enzyme before substrate addition [1].
The inhibitory potency (IC50) of compounds against PFKFB3 and PFKFB4 was determined using this assay format [1].
Isothermal Titration Calorimetry (ITC) was performed by titrating KAN0438241 (200 μM) into PFKFB3 protein (20 μM) to confirm binding and determine affinity [1].

Clonogenic Survival Assay: U2OS, BJ TERT, or BJ RAS cells were seeded at low density. For siRNA experiments, cells were transfected, irradiated 24h later, and seeded for colony formation. For inhibitor studies, cells were treated with vehicle or KAN0438757 for 24h, then irradiated. Inhibitors were washed out after a specified time (72h for inhibitor studies, 4h for siRNA studies). Colonies were allowed to form for several days (4-12 days depending on cell line), then fixed, stained with methylene blue, and counted manually [1].
Immunofluorescence Microscopy and Foci Quantification: Cells grown on coverslips were treated and/or irradiated. For visualizing repair foci proteins like PFKFB3, RPA32, RAD51, γH2AX, and RRM2, cells were subjected to in situ cell fractionation (extraction with a cytoskeleton buffer containing detergent) prior to fixation to reduce background. Cells were then fixed, permeabilized, blocked, and incubated with primary and fluorescent secondary antibodies. DNA was stained with DAPI. Images were acquired by confocal microscopy. Foci number, intensity, and co-localization were quantified using CellProfiler software [1].
DR-GFP Homologous Recombination Repair Assay: U2OS DR-GFP reporter cells were treated with siRNA or inhibitors. 24h later, cells were transfected with an I-SceI endonuclease expression vector to induce a site-specific double-strand break. 24-48h after transfection, cells were harvested, and the percentage of GFP-positive cells (indicating successful HR repair) was measured by flow cytometry. HR activity was calculated relative to control-treated cells [1].
EdU Incorporation Assay (Repair Synthesis): U2OS cells were treated, irradiated, and pulsed with EdU for 30 min at various recovery times. Cells were fixed, and incorporated EdU was detected via a click chemistry-based fluorescent conjugation. DNA content was stained with Hoechst. Cells in G2/M phase (based on DNA content) that were EdU-positive were quantified by flow cytometry to specifically assess nucleotide incorporation during repair, excluding S-phase replication [1].
EdU Incorporation Assay (Replication): U2OS cells were treated with compounds for several hours, pulsed with EdU for the last 40 min, then fixed. Incorporated EdU was detected via click chemistry and fluorescence intensity per cell was quantified using image analysis software (CellProfiler) [1].
DNA Fiber Assay: U2OS cells were treated with inhibitors for 24h. Replication forks were sequentially labeled with two different halogenated nucleotides (CldU for 20 min, then IdU for 20 min). Cells were harvested, and DNA fibers were spread on slides, fixed, and denatured. Incorporated nucleotides were detected with specific antibodies. The length of IdU and CldU tracks was measured using ImageJ software, and fork speed (in kilobases per minute) was calculated [1].
Intracellular dNTP Measurement: dNTP levels were measured using an HIV-1 reverse transcriptase-based assay. Cells were trypsinized, counted, and dNTPs were extracted with methanol. The dried extracts were resuspended, and a primer extension reaction was performed by HIV-1 RT in the presence of a specific primer/template and one dNTP. The amount of extended product, corresponding to the level of that particular dNTP in the extract, was quantified. Amounts were normalized to cell number and related to control treatment [1].
Cellular Thermal Shift Assay (CETSA): U2OS cells were treated with KAN0438757 or DMSO, harvested, and lysed by freeze-thaw cycles. Cell lysates were aliquoted, heated to a range of temperatures, and centrifuged to remove precipitated proteins. The remaining soluble protein in the supernatant was quantified and analyzed by western blot for PFKFB3. Stabilization of PFKFB3 (indicating ligand binding) was assessed by comparing its melting curve in drug-treated vs. vehicle-treated samples [1].
Van Schaftingen Assay (Intracellular F-2,6-P2): Cancer cells were starved in low-glucose/low-serum medium overnight, then treated with compounds and stimulated with glucose. Cells were lysed with NaOH, and the lysates were neutralized. F-2,6-P2 levels were quantified using a coupled enzymatic assay based on the potent activation of pyrophosphate-dependent phosphofructokinase-1 (PPi-PFK) by F-2,6-P2. The activation leads to consumption of NADH, which is monitored spectrophotometrically. The decrease in absorbance is proportional to F-2,6-P2 concentration within a linear range [1].

In the process of drug discovery, the properties of compounds are characterized, such as buffer stability, solubility, metabolic stability, plasma stability, protein binding and permeability[1]. Specifically, the prodrug design of KAN0438757 (an ester of the active acid KAN0438241) was designed to improve cell permeability and achieve efficient intracellular hydrolysis to release the active compound[1].

Unlike cancer cell lines, the viability of peripheral blood mononuclear cells (PBMCs) was not affected by the inhibition of PFKFB3 by KAN0438757[1]. In clonogenic assays, untransformed BJ TERT cells showed only a slight decrease in survival at KAN0438757 concentrations, while the survival of transformed BJ RAS cells was significantly reduced, especially when used in combination with IR, suggesting a potential therapeutic window[1].

[1]. Targeting PFKFB3 radiosensitizes cancer cells and suppresses homologous recombination. Nat Commun. 2018 Sep 24;9(1):3872.

[2]. Effects of the Novel PFKFB3 Inhibitor KAN0438757 on Colorectal Cancer Cells and Its Systemic Toxicity Evaluation In Vivo. Cancers (Basel). 2021 Feb 28;13(5):1011.

KAN0438757 is a selective small molecule glycolytic enzyme PFKFB3 inhibitor developed to study the role of PFKFB3 in DNA repair [1]. This study reveals a new, non-classical role of PFKFB3 in DNA damage response. PFKFB3 is recruited to DNA double-strand break sites via the MRN-ATM-γH2AX-MDC1 pathway. Its enzymatic activity is essential for the subsequent recruitment of the RRM2 subunit of ribonucleotide reductase and HR repair factors (RPA, RAD51), thereby promoting the production and repair synthesis of local dNTPs [1]. Inhibition of PFKFB3 with KAN0438757 impairs homologous recombination repair, reduces the cellular dNTP pool, halts replication forks, and makes cancer cells sensitive to ionizing radiation, while not affecting non-transformed cells. This makes PFKFB3 inhibition a potential strategy for cancer-specific radiosensitization [1].
The previously reported PFKFB3 inhibitor 3-PO did not show activity in the authors’ enzymatic or cellular HR repair assays, prompting the development of KAN0438757[1].
The cocrystal structure of the active metabolite KAN0438241 with human PFKFB3 shows that it binds in a fructose-6-phosphate substrate pocket, mimicking the interaction of endogenous substrates, and is a non-ATP-competitive inhibitor[1].

C21H18FNO7S

447.433528423309

447.078

1451255-59-6

71586631

Typically exists as solid at room temperature

3.2

4

9

8

31

700

0

QRDFCYMUXHUTCS-UHFFFAOYSA-N

InChI=1S/C21H18FNO7S/c22-14-4-7-19(25)18(11-14)13-2-1-3-16(10-13)31(28,29)23-15-5-6-17(20(26)12-15)21(27)30-9-8-24/h1-7,10-12,23-26H,8-9H2

2-hydroxyethyl 4-[[3-(5-fluoro-2-hydroxyphenyl)phenyl]sulfonylamino]-2-hydroxybenzoate

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 : ~130 mg/mL (~290.55 mM)

Solubility in Formulation 1: 2.17 mg/mL (4.85 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 21.7 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: ≥ 2.17 mg/mL (4.85 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 21.7 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: ≥ 2.17 mg/mL (4.85 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 21.7 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.)