PLK1 (IC50 = 2.2 nM); PLK3 (IC50 = 9.1 nM); PDGFR1β (IC50 = 160 nM); VEGFR2 (IC50 = 360 nM); Aurora A (IC50 = 4800 nM); CDK2/cyclin A (IC50 = 7600 nM)
Compound 1 is a potent and selective ATP-competitive inhibitor of Polo-like kinase 1 (PLK1) and PLK3. The reported IC₅₀ values are 2.2 nmol/L for PLK1 and 9.1 nmol/L for PLK3. It is >100-fold selective for PLKs over other kinases tested (e.g., CDK2/cyclin A, CDK1/cyclin A, Aurora A, PDGFR1β, VEGFR2, p38α, p38β, EGFR, c-src).[1]

GW843682X (compound 1) is effectively suppresses the growth of tumor cells, as demonstrated by IC50 values of 0.41, 0.57, 0.11, 0.38, and 0.70 μM for HeLa, A549, H460, and HCT116 cell lines. With an IC50 of 0.14 μM, GW843682X dose-dependently inhibits PLK1 phosphorylation of Ser15-p53. GW843682X (3 μM) treatment for 24, 48, and 72 hours results in a strong G2-M arrest in HDF cells and H460 cells. GW843682X (5 μM) causes H460 cells to undergo apoptosis as opposed to HDF cells[1]. At a 120 nM EC50, GW843682X suppresses the growth of U937 cells. U937 cells' 50% mitotic entry is suppressed when GW843682X (500 nM) and 5 µM VP-16 are combined[2]. Inhibiting the growth of acute leukemia cells, GW843682X (0.06-1μM) enhances vincristine's anti-proliferative qualities. Leukemia cells undergo apoptosis when exposed to GW843682X (0.1–1 μM) in a manner that is dependent on both time and dose. Bcl-xl is dephosphorylated in leukemia cells by GW843682X (0.5–1 μM)[3].

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Compound 1 potently inhibits the proliferation of a wide variety of human tumor cell lines in culture, with submicromolar IC₅₀ values ranging from 0.11 to 0.70 μmol/L for most lines (e.g., HeLa: 0.11 μmol/L, H460: 0.38 μmol/L, HCT116: 0.70 μmol/L). A notable exception is the PC-3 prostate carcinoma cell line (IC₅₀ = 6.82 μmol/L).[1]
Compared to normal human diploid fibroblasts (HDF, IC₅₀ = 6.14 μmol/L), compound 1 shows >10-fold selectivity for tumor cells, suggesting a potential therapeutic window.[1]
Compound 1 is equipotent against the P-glycoprotein-overexpressing, multidrug-resistant uterine sarcoma cell line MES-SA/DX5 (IC₅₀ ~0.21 μmol/L) and its drug-sensitive parental line MES-SA, indicating it is not a substrate for the P-glycoprotein efflux pump.[1]
Treatment of H460 lung adenocarcinoma cells with compound 1 induces a transient G₂-M phase arrest, followed by apoptosis, as evidenced by increased sub-2N DNA content, activation of caspase-3, and cleavage of PARP.[1]
In contrast, normal human diploid fibroblasts (HDF) treated with compound 1 undergo a strong G₂-M arrest with minimal apoptosis over 72 hours.[1]
Immunofluorescence microscopy in H460 cells treated with compound 1 (0.5-1 μmol/L) reveals phenotypes consistent with PLK1 inhibition, including aberrant mitotic spindles at 24 hours and multinucleated cells at 48 hours.[1]
A cellular mechanistic assay using a tetracycline-inducible HeLa cell line expressing a constitutively active p53-PLK1(T210D) fusion protein showed that compound 1 inhibits intracellular PLK1 kinase activity with an IC₅₀ of 0.14 μmol/L, as measured by the reduction in phosphorylation of p53 at Ser15.[1]

Trichoplusia ni cells infected with baculoviruses are used to prepare PLK1 and PLK3 proteins. The following is the method used to determine PLK1 and PLK3 enzyme activity. Every measurement was made in an environment where the amount of signal produced rose linearly with the enzyme and time. Test compounds are added at varying known concentrations in 100% DMSO to white 384-well assay plates (0.1 μL for 10 μL and some 20 μL assays, 1 μL for some 20 μL assays). As controls, DMSO (1-5% final) and EDTA (65 mM) are employed. The components of the reaction mix at 22°C are as follows: pH 7.2 HEPES with a concentration of 25 mM, 15 mM MgCl₂, 1 μM ATP, 0.05 μCi/well [γ-³³P]ATP (10 Ci/mmol), 1 μM substrate peptide (Biotin-Ahx-SFNDTLDFD), 0.15 mg/mL bovine serum albumin, 1 mM DTT, and either 2 nM PLK1 kinase domain or 5 nM full-length PLK3. Using automated liquid handlers, quickly add 10 or 20 μL of Reaction Mix to each well after adding the enzyme, and then incubate for 1 to 1.5 hours at 22°C. Using 50 μL of stop mix [50 mM EDTA, 4.0 mg/mL streptavidin SPA beads in Dulbecco's PBS (without Mg²⁺ and Ca²⁺), 50 μM ATP] per well, the 20 μL enzymatic reactions are terminated. Ten microliters (50 microliters) of stop mix—three milliliters of streptavidin-coupled SPA Imaging beads in Dulbecco's PBS (without Mg²⁺ and Ca²⁺) and fifty microliters of EDTA—per well are used to halt the ten microliter reactions. The plates are either sealed, spun at 500 × g for a minute or left to settle overnight, and then counted in Packard TopCount for 30 seconds per well or imaged using a Viewlux imager. In comparison to the results obtained in control (DMSO-only) wells, the signal above background (EDTA controls) is converted to a percent inhibition[1].

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PLK1 and PLK3 enzyme inhibition assays were performed using recombinant proteins. Test compounds were dispensed in DMSO into white 384-well plates. The reaction mixture, containing HEPES buffer (pH 7.2), MgCl₂, ATP (including trace [γ-³²P]ATP), a biotinylated substrate peptide (Biotin-Ahx-SFNDTLDFD), bovine serum albumin, DTT, and either the PLK1 kinase domain or full-length PLK3, was added to initiate the reaction. After incubation at 22°C for 1 to 1.5 hours, reactions were stopped with a solution containing EDTA and streptavidin-coated scintillation proximity assay (SPA) beads. Plates were sealed, centrifuged or settled, and radioactivity was measured using a microplate scintillation counter. Inhibition was calculated relative to DMSO (vehicle) and EDTA (negative) controls.[1]

Data analysis and experimentation are done. Specifically, in these assays, 2,000 H460 cells are plated per well, 5,000 HDF cells are plated per well, and 7,000 and 6,000 per well, respectively, are plated in a 96-well plate for the drug-resistant cell line MES-SA/DX5 and its sensitive parent line MES-SA. Throughout the three days of the experiment, these densities enabled the vehicle controls to grow logarithmically. The compound (30-0.00152 μM) is subjected to threefold dilutions in three different media: low-glucose DMEM, which contains 5% FBS, 50 μg/mL gentamicin, and 0.3% (v/v) DMSO (HDF cells); RPMI 1640, which contains 5% FBS, 50 μg/mL gentamicin, and 0.3% (v/v) DMSO (H460); or McCoy's 5A, which contains 5% FBS, 50 μg/mL gentamicin, and 0.3% (v/v) DMSO (MES-SA and MES-SA/DX5)[1].

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For cell proliferation inhibition assays, cells (e.g., H460, HDF) were seeded in 96-well plates at specified densities. After attachment, cells were exposed to serial 3-fold dilutions of compound 1 (0.00152 to 30 μmol/L) in medium containing 0.3% DMSO for 72 hours. Cell viability was then assessed using a methylene blue staining and absorbance measurement protocol.[1]
For cell cycle analysis by flow cytometry, H460 and HDF cells were treated with 1 or 3 μmol/L compound 1 for 24, 48, and 72 hours. Cells (including floating cells) were collected, fixed in ethanol, permeabilized, stained with propidium iodide for DNA content, and analyzed using a flow cytometer.[1]
Apoptosis was assessed by measuring caspase-3 activity. H460 cells treated with 5 μmol/L compound 1 over time were lysed, and the lysates were incubated with the fluorogenic caspase-3 substrate AC-DEVD-AMC. Cleavage of the substrate was monitored fluorometrically.[1]
PARP cleavage was analyzed by Western blot. Lysates from H460 cells treated with 5 μmol/L compound 1 were subjected to SDS-PAGE, transferred to nitrocellulose, and probed with an antibody that recognizes both full-length (116 kDa) and cleaved (85 kDa) PARP.[1]
For immunofluorescence microscopy, H460 cells grown on chamber slides were treated with 0.5 or 1 μmol/L compound 1 for 24 or 48 hours, fixed with methanol, permeabilized, and stained with antibodies against α-tubulin (FITC-conjugated), lamin A/C, and DAPI. Images were captured using a fluorescence microscope.[1]
The cellular mechanistic assay for intracellular PLK1 inhibition utilized a HeLa Tet-On cell line engineered to express a tetracycline-inducible, biotinylated p53-PLK1(T210D) fusion protein. Cells were induced with doxycycline, treated with compound 1 for 1 hour, and lysed. The lysates were transferred to anti-p53 antibody-coated plates, and the level of p53 phosphorylated at Ser15 was quantified by ELISA using a phospho-specific antibody and HRP detection.[1]

[1]. In vitro biological activity of a novel small-molecule inhibitor of polo-like kinase 1. Mol Cancer Ther. 2007 Feb;6(2):450-9. Epub 2007 Jan 31.

[2]. Evaluation of Polo-like Kinase 1 inhibition on the G2/M checkpoint in Acute Myelocytic Leukaemia. Eur J Pharmacol. 2008 Sep 4;591(1-3):102-5.

[3]. A novel treatment strategy targeting polo-like kinase 1 in hematological malignancies. Leukemia. 2009 Sep;23(9):1564-76.

Compound 1 is a novel thiophene benzimidazole ATP-competitive inhibitor discovered for its potential as an anticancer drug. It targets PLK1, a kinase crucial for mitosis that is frequently overexpressed in tumors. [1]
This study also developed a novel cellular mechanism detection method (p53-PLK1 fusion protein ELISA) to directly measure intracellular PLK1 inhibition. This method showed a good correlation with antiproliferative activity, validating the target mechanism of the compound. [1]
Compound 1 can also effectively inhibit another member of the PLK family, PLK3, making it a useful tool molecule for studying the biological functions of PLK1 and PLK3. [1]

C22H18N3O4F3S

477.45622

477.10

C, 55.34; H, 3.80; F, 11.94; N, 8.80; O, 13.40; S, 6.71

660868-91-7

660868-91-7

9826308

Light yellow to yellow solid powder

1.4±0.1 g/cm3

629.6±65.0 °C at 760 mmHg

334.6±34.3 °C

0.0±1.8 mmHg at 25°C

1.613

4.25

1

9

7

33

688

0

O=C(C1=C(OCC2=CC=CC=C2C(F)(F)F)C=C(N3C4=CC(OC)=C(OC)C=C4N=C3)S1)N

JSKUWFIZUALZLX-UHFFFAOYSA-N

InChI=1S/C22H18F3N3O4S/c1-30-16-7-14-15(8-17(16)31-2)28(11-27-14)19-9-18(20(33-19)21(26)29)32-10-12-5-3-4-6-13(12)22(23,24)25/h3-9,11H,10H2,1-2H3,(H2,26,29)

5-(5,6-dimethoxybenzimidazol-1-yl)-3-[[2-(trifluoromethyl)phenyl]methoxy]thiophene-2-carboxamide

GW843682X; GW-843682X; GW 843682X

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: 33.3~95 mg/mL (69.8~199 mM)
Ethanol: ~3 mg/mL (~6.3 mM)

Solubility in Formulation 1: 2.5 mg/mL (5.24 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 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 (5.24 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.)