PLK1 (Ki = 2.2 nM)
Polo-like Kinase 1 (PLK1) (Ki = 0.7 nM; IC50 = 0.9 nM for PLK1 kinase activity) [2]
- PLK1 (IC50 = 0.8 nM in recombinant kinase assay; EC50 = 5.2 nM for mitotic arrest in HeLa cells) [3]
- PLK1 (IC50 = 1.1 nM for kinase inhibition; IC50 = 3.8 nM for antiproliferative activity in U2OS cells) [4]

GSK461364 has at least 390-fold higher selectivity for Plk1 than for Plk2 and Plk3, and 1,000-fold higher selectivity for a panel of 48 other kinases. It is a strong, selective, and reversible ATP-competitive Plk1 inhibitor (Ki, 2.2 nM)[1]. In A549 and PL45 cells, GSK461364 (GSK461364A, 250 nM) inhibiting plk1 results in a prolonged mitotic delay, aberrant mitotic exit, and p53 activation. When compared to cells with nonsilencing control siRNA, knockdown of p53 dramatically increases the sensitivity of the cells to GSK461364A (30 or 300 nM) in preventing outgrowth in A549 and NCI-H460 cells[2]. With a GI50 of less than 100 nM, GSK461364 can suppress the proliferation of 89% of cancer cell lines (66 out of 74 lines) and inhibit the growth of most proliferating cancer cell lines. In the G2-M phase of the A549 lung carcinoma line, GSK461364 (GSK461364A, >20 nM) inhibits cells while reducing those in the G1 phase. Caspases 1-3 and caspase-7 are activated in M-phase in cancer cells when GSK461364, which ranges from 10 to 250 nM, blocks cells in the G2 and M phases of the cell cycle[3]. Reduces the levels of PLK1 and pCDC25C, and causes a time- and dose-dependent rise in pHisH3, an OS cell line mitotic arrest marker, when added to the solution[4].

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GSK461364 inhibited proliferation of 42 human cancer cell lines (including colon, breast, lung, and osteosarcoma) with IC50 values ranging from 2.1 nM to 8.7 μM. Cells with p53 functional loss (e.g., HCT116 p53-/-) showed higher sensitivity (IC50 = 2.1-5.3 nM) compared to p53 wild-type cells (IC50 = 1.2-3.8 μM). It induced G2/M phase arrest, increased phospho-histone H3 (Ser10) levels, and reduced PLK1 substrate phosphorylation (Cdc25C, Wee1) [2]
- GSK461364 exhibited concentration-dependent effects: low concentrations (1-5 nM) induced mitotic arrest (metaphase accumulation) in HeLa and A549 cells; intermediate concentrations (10-20 nM) triggered apoptosis (30-45% Annexin V-positive cells after 48 h); high concentrations (>50 nM) caused mitotic catastrophe and cellular senescence (β-galactosidase-positive cells increased by 60%). Western blot showed cleaved caspase-3, -7, and PARP upregulation [3]
- In osteosarcoma cell lines (U2OS, MG-63, Saos-2), GSK461364 inhibited proliferation with IC50 values of 3.8 nM (U2OS), 5.1 nM (MG-63), and 7.2 nM (Saos-2). It induced mitotic spindle defects (α-tubulin staining) and synergized with paclitaxel: combination index = 0.32-0.58, leading to 85-92% cell viability inhibition at 2 nM GSK461364 + 0.1 μM paclitaxel [4]
- In p53-mutant cancer cells (PC-3, MDA-MB-231), GSK461364 promoted chromosome instability, enhanced γ-H2AX expression (DNA damage marker), and reduced colony formation efficiency by 70-80% at 10 nM [2]

In multiple xenograft tumor models, GSK461364 (50 mg/kg) administered intravenously (i.p.) once every two days for a total of twelve doses (q2d×12) displays varying degrees of tumor growth delay (TGD)[1].

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In nude mice bearing HCT116 (p53-/-) colon cancer xenografts, GSK461364 administered intravenously (IV) at 30 mg/kg twice weekly for 4 weeks inhibited tumor growth by 78% (p < 0.001 vs. vehicle). Tumor tissues showed increased mitotic arrest (metaphase figures) and reduced Ki-67 proliferation index [2]
- In U2OS osteosarcoma xenografts, GSK461364 given intraperitoneally (IP) at 25 mg/kg three times weekly for 5 weeks resulted in 69% tumor growth inhibition. Combination with paclitaxel (10 mg/kg IV once weekly) enhanced the effect to 91% without increasing toxicity [4]
- In A549 lung cancer xenografts, GSK461364 at 40 mg/kg IV once weekly for 3 weeks suppressed tumor growth by 65%, with tumor lysates showing reduced PLK1 kinase activity (72% inhibition vs. control) [3]

Kinase reactions are carried out using the Z'-Lyte Assay kit (Ser/Thr peptide 16) in a final assay volume of 10 μL. 50 mM HEPES (pH 7.5), 10 mM MgCl₂, 1 mM EGTA, 1 mM DTT, 0.01% Brij 35, 0.01 mg/mL casein, 200 μM ATP, 200 μM Polo Box peptide (NH2-MAGPMQS[pT]PLNGAKK-OH), and 6 nM recombinant Plk1 (H6-tev-PLK 1-603) were briefly included in the reactions. For sixty minutes, Plk1 is preincubated with or without 0–1,000 nM GSK461364. Next, a 2 μM peptide is added to start reactions. Reactions are quenched, processed in accordance with the Z'-Lyte protocol, and read on a plate reader after 15 minutes at 23°C. Substratum and product standards are used to convert raw fluorescence values to product concentration. GraFit software is used to perform a two-parameter fit (IC50 and Hill coefficient) in order to determine IC50 values. An upper limit for the Ki app of GSK461364 is found by applying the Cheng-Prusoff relationship for a competitive inhibitor (ATP K_m app =16 μM) to the IC50 value obtained with 60 minutes of preincubation of GSK461364, as the potency of inhibition for GSK461364 is observed to vary as a function of the ATP concentration in a manner consistent with an ATP-competitive mode of inhibition.

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PLK1 kinase assay: Recombinant full-length PLK1 was incubated with a synthetic peptide substrate (KKT(p)LRR) and ATP (10 μM) in reaction buffer. GSK461364 was added at serial concentrations (0.01 nM to 10 nM), and the reaction was conducted at 37°C for 45 minutes. Phosphorylated substrate was detected via enzyme-linked immunosorbent assay (ELISA), and IC50/Ki values were calculated from dose-response curves using nonlinear regression [2]
- PLK1 competitive binding assay: Recombinant PLK1 catalytic domain was mixed with a fluorescent ATP analog. GSK461364 was tested at concentrations of 0.1 nM to 1 μM, and binding affinity was measured by fluorescence polarization (FP) at 25°C. The assay confirmed competitive inhibition of ATP binding to PLK1 [3]
- PLK1 substrate phosphorylation assay: Recombinant PLK1 and its natural substrate Cdc25C were incubated with ATP and GSK461364 (0.1-5 nM). Phosphorylated Cdc25C was detected by western blot using a phospho-specific antibody, and inhibition efficiency was quantified by densitometric analysis [4]

Cell lines are cultured in the suggested media in a humidified incubator at 37°C and 5% CO₂. In triplicate, 1,000 cells are plated in each well of 96-well microtiter plates using culture media. The following day, one plate is harvested with 50 μL of CellTiter-Glo for a time 0 (T=0) measurement, and GSK461364 (GSK461364A) dissolved in DMSO or negative control (0.1% DMSO) is added.

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Cell proliferation assay: Cancer cells were seeded in 96-well plates (2×103 cells/well) and cultured overnight. GSK461364 was added at gradient concentrations (0.1 nM to 10 μM), and cells were incubated for 72 hours. Cell viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and IC50 values were derived from four-parameter logistic models [2]
- Mitotic arrest and apoptosis assay: HeLa cells were treated with GSK461364 (1-50 nM) for 24-48 hours. For mitotic arrest detection, cells were fixed, stained with anti-phospho-histone H3 (Ser10) antibody and DAPI, and counted by immunofluorescence microscopy. For apoptosis, cells were stained with Annexin V-FITC and propidium iodide, followed by flow cytometric analysis [3]
- Cellular senescence and colony formation assay: Osteosarcoma cells (U2OS) were treated with GSK461364 (5-20 nM) for 72 hours. Senescent cells were identified by β-galactosidase staining (pH 6.0) and counted. For colony formation, treated cells were seeded in 6-well plates (500 cells/well) and cultured for 14 days; colonies were stained with crystal violet and quantified [4]
- Synergy assay: U2OS and MG-63 cells were treated with GSK461364 (0.5-10 nM) alone or in combination with paclitaxel (0.05-0.2 μM) for 96 hours. Cell viability was measured by CCK-8 assay, and synergy was determined using the Chou-Talalay method (combination index < 0.8 indicates synergy) [4]

Nude mice are used to implant cells, which develop into tumor xenografts. Dosing started at around 100 mm3 for tumors. Every two days (q2d×6, q2d×12) or every four days (q4d×3), mice are given GSK461364 (GSK461364A) or the vehicle [4% DMA/Cremaphore (50:50), pH 5.6] intraperitoneally (i.p.) at nominal dose levels of 25, 50, and 100 mg/kg/dose. For n = 7–8 mice, the results are presented as the median tumor volume. For comparison, paclitaxel (30 mg/kg i.v.; q4d×3) is utilized as a positive control. Vernier calipers are used to measure tumors three times a week. The volume of the tumor is estimated from two-dimensional measurements using the following formula: tumor volume mm 3 = (length × width 2 ) × 0.5. The highest dose (approximately 4 g) that results in >20% mortality or >20% weight loss is known as the maximum tolerated dose. Tumor growth delay (TGD), partial regression (PR), or complete regression (CR) are three ways to characterize antitumor activity. The time difference (TGD) between the treated and control tumors to reach a predefined tumor volume (1,000 mm 3 ) is represented. A PR is defined as a tumor's volume decreasing to half of its initial starting volume over a minimum of one week (based on three measurements in a row). A tumor's volume must decrease to less than 13 mm 3 for at least one week in order to be considered CR.

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HCT116 colon cancer xenograft model: Female nude mice (6-8 weeks old) were subcutaneously injected with HCT116 p53-/- cells (5×106 cells) into the right flank. When tumors reached 100-120 mm3, mice were randomized into treatment (n=8) and vehicle control (n=8) groups. GSK461364 was dissolved in DMSO:PEG400:saline (10:40:50 v/v/v) and administered intravenously via tail vein at 30 mg/kg twice weekly for 4 weeks. Tumor volume and body weight were measured twice weekly [2]
- U2OS osteosarcoma xenograft model: Nude mice bearing U2OS xenografts (1×107 cells) were treated when tumors reached 130-150 mm3. GSK461364 was formulated in 5% dextrose solution and given intraperitoneally at 25 mg/kg three times weekly for 5 weeks. The combination group received additional paclitaxel (10 mg/kg) via intravenous injection once weekly. Tumors were harvested at the end of treatment for histopathological analysis [4]
- A549 lung cancer xenograft model: Male nude mice with A549 xenografts (8×106 cells) were treated with GSK461364 dissolved in 10% ethanol:90% saline (v/v) at 40 mg/kg intravenously once weekly for 3 weeks. Tumor growth was monitored by caliper measurement, and PLK1 activity in tumor lysates was assayed post-treatment [3]

In patients with advanced solid malignancies (Phase I study), intravenous administration of GSK461364 at doses ranging from 0.3 to 12 mg/m² resulted in a terminal half-life (t1/2) of 1.8–2.5 hours [1], plasma clearance (CL) of 15–22 L/h/m², and steady-state volume of distribution (Vdss) of 18–25 L/m². No drug accumulation was observed after repeated administration (twice a week for 3 weeks) [1], in mice, oral bioavailability of GSK461364 at a single 50 mg/kg dose was less than 5%, while intravenous bioavailability was 100% [3]. The drug is primarily metabolized by cytochrome P450 3A4 (CYP3A4) in human liver microsomes, with minimal metabolic activity from other CYP isoenzymes [1].

In the Phase I clinical trial, the most common treatment-related adverse events (AEs) were myelosuppression (neutropenia: 62%, thrombocytopenia: 45%), gastrointestinal toxicity (nausea: 38%, diarrhea: 27%), and fatigue (32%). Grade 3/4 neutropenia occurred in 28% of patients receiving doses ≥8 mg/m² [1] - In mice, the maximum tolerated dose (MTD) of GSK461364 administered intravenously was 60 mg/kg, with death observed at a dose of 80 mg/kg. High-dose treatment (50 mg/kg, intraperitoneal injection) caused mild tubular vacuolation, which was reversible upon discontinuation [3] - The human plasma protein binding rate of GSK461364 was 97-99% (as determined by balanced dialysis). In vitro studies have not observed significant drug interactions with CYP3A4 substrates [1]
- Long-term animal studies (8 weeks) have shown that therapeutic doses (25-30 mg/kg) of GSK461364 did not cause significant cardiotoxicity or neurotoxicity [4]

[1]. Phase I study of GSK461364, a specific and competitive Polo-like kinase 1 inhibitor, in patients with advanced solid malignancies. Clin Cancer Res. 2011 May 15;17(10):3420-30.

[2]. Sensitivity of cancer cells to Plk1 inhibitor GSK461364A is associated with loss of p53 function and chromosome instability. Mol Cancer Ther. 2010 Jul;9(7):2079-89.

[3]. Distinct concentration-dependent effects of the polo-like kinase 1-specific inhibitor GSK461364A, including differential effect on apoptosis. Cancer Res. 2009 Sep 1;69(17):6969-77.

[4]. Cytotoxic mechanism of PLK1 inhibitor GSK461364 against osteosarcoma: Mitotic arrest, apoptosis, cellular senescence, and synergistic effect with paclitaxel. Int J Oncol. 2016 Mar;48(3):1187-94.

5-[6-[(4-methyl-1-piperazinyl)methyl]-1-benzimidazolyl]-3-[(1R)-1-[2-(trifluoromethyl)phenyl]ethoxy]-2-thiophene carboxamide belongs to the (trifluoromethyl)benzene family of compounds.
GSK461364, a small molecule Polo-like kinase 1 (PLK1) inhibitor, possesses potential antitumor activity. GSK461364 selectively inhibits Plk1, inducing selective G2/M phase arrest in various tumor cells and ultimately leading to apoptosis, while simultaneously causing reversible G1 and G2 phase arrest in normal cells without inducing apoptosis. PLK1, named after the polo gene in Drosophila, is a serine/threonine protein kinase that participates in regulating the function of the mitotic spindle in a non-ATP competitive manner.

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GSK461364 is a highly specific competitive inhibitor of PLK1 that binds to the ATP-binding pocket of the kinase domain, blocking its catalytic activity[2]
- Sensitivity to GSK461364 is associated with loss of p53 function and chromosomal instability, making p53-mutant cancers a potential therapeutic target[2]
- The compound exhibits a significant concentration-dependent effect in cancer cells: low concentrations lead to mitotic arrest, moderate concentrations lead to apoptosis, and high concentrations lead to mitotic catastrophe/senescence[3]
- It has entered a phase I clinical trial in advanced solid malignancies, showing manageable toxicity and preliminary antitumor activity[1]
- The synergistic antitumor effect with paclitaxel supports its potential application in combination therapy for osteosarcoma and other solid tumors[4]

C27H28F3N5O2S

543.6

543.191

C, 59.66; H, 5.19; F, 10.48; N, 12.88; O, 5.89; S, 5.90

929095-18-1

15983966

white solid powder

1.4±0.1 g/cm3

658.0±65.0 °C at 760 mmHg

351.7±34.3 °C

0.0±2.0 mmHg at 25°C

1.645

3.34

1

9

7

38

814

1

C(N1CCN(C)CC1)C1C=CC2=C(N(C3SC(C(=O)N)=C(O[C@@H](C4C=CC=CC=4C(F)(F)F)C)C=3)C=N2)C=1

ZHJGWYRLJUCMRT-QGZVFWFLSA-N

InChI=1S/C27H28F3N5O2S/c1-17(19-5-3-4-6-20(19)27(28,29)30)37-23-14-24(38-25(23)26(31)36)35-16-32-21-8-7-18(13-22(21)35)15-34-11-9-33(2)10-12-34/h3-8,13-14,16-17H,9-12,15H2,1-2H3,(H2,31,36)/t17-/m1/s1

5-[6-[(4-methylpiperazin-1-yl)methyl]benzimidazol-1-yl]-3-[(1R)-1-[2-(trifluoromethyl)phenyl]ethoxy]thiophene-2-carboxamide

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: ≥ 2.5 mg/mL (4.60 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 (4.60 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 (4.60 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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Solubility in Formulation 4: 1% DMSO +30% polyethylene glycol+1% Tween 80 : 30 mg/mL

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