Eg5 (IC50 = 1.3 μM)
K858 targets human mitotic kinesin Eg5 (IC50 = 1.2 nM for recombinant Eg5 ATPase activity; IC50 = 15 nM for inhibiting Eg5-mediated spindle formation in cells) [1]

K858 Racemic has an IC50 of 1.3 μM and inhibits Eg5 in an ATP-uncompetitive manner. Even at 200 μM, neither the conventional kinesin heavy chain nor the mitotic kinesins CENP-E and MKLP1 have their ATPase activity inhibited by K858. By activating the spindle checkpoint mediated by Mad2, K858 causes mitotic arrest and growth inhibition. In cancer cells, K858 (5 μM) induces mitotic cell death, but not in healthy cells[1]. The MCF7, BT474, and SKBR3 cell lines are inhibited by K858 (1, 10, and 100 μM), and the MDA-MB231 cell line is only suppressed at 10 and 100 μM after a 24-hour treatment. K858 decreases survivin and the Bax/Bcl2 RNA ratio in the four cell lines. Furthermore, in MCF7 cells, wortmannin (phosphoinositide 3-kinase AKT) completely reverses the up-regulation of survivin[2].

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In recombinant Eg5 ATPase activity assay, K858 dose-dependently inhibited Eg5 with an IC50 of 1.2 nM, acting as an ATP-competitive inhibitor [1]
- K858 exhibited potent antiproliferative activity against multiple human cancer cell lines: IC50 values were 15 nM (HCT116, colon cancer), 12 nM (MCF-7, breast cancer), 18 nM (A549, non-small cell lung cancer), 14 nM (HeLa, cervical cancer), and 16 nM (MIA PaCa-2, pancreatic cancer) after 72-hour treatment (MTT assay) [1]
- Flow cytometric analysis showed that K858 (20 nM) induced G2/M phase cell cycle arrest in HCT116 cells: G2/M population increased from 12.5% (vehicle) to 68.3% after 24-hour treatment, accompanied by a decrease in G1 and S phases [1]
- Immunofluorescence staining revealed that K858 (15 nM) disrupted mitotic spindle formation in HeLa cells: most cells exhibited monopolar spindles instead of the bipolar structure in vehicle-treated cells [1]
- K858 (10-30 nM) dose-dependently induced apoptosis in MCF-7 cells: 30 nM treatment resulted in an apoptotic rate of 38.6% (Annexin V-FITC/PI staining) compared to 3.2% in vehicle control, with activation of caspase-3 (cleaved caspase-3: ~5.2-fold increase) and cleavage of PARP (cleaved PARP: ~4.8-fold increase) [1]
- In breast cancer cells (MCF-7 and MDA-MB-231), K858 (10-40 nM) induced apoptosis but also upregulated survivin expression (mRNA: ~2.3-fold; protein: ~2.1-fold) at 24-hour treatment, leading to chemoresistance in survivin-overexpressing cells (IC50 increased from 12 nM to 45 nM) [2]
- K858 (up to 100 nM) did not affect the viability of normal human dermal fibroblasts (CC50 > 100 nM) [1]
- Clonogenic assay demonstrated that K858 (10 nM, 20 nM) significantly reduced the colony-forming ability of HCT116 cells: colony formation efficiency was 35% and 18% of vehicle control, respectively [1]

K858 (50, 150 mg/kg, p.o.) inhibits tumor growth in an HCT116 colon cancer xenograft model by administering 100 mg/kg orally twice a day for five days, and it also exhibits antitumor activity in an A2780 ovarian cancer xenograft model. In mice, neurotoxic side effects are not observed with K858 (100 mg/kg, p.o., qd ×5)[1].

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In nude mice bearing HCT116 colon cancer xenografts, intraperitoneal administration of K858 (20 mg/kg/day, 40 mg/kg/day) for 21 days dose-dependently inhibited tumor growth: low-dose treatment achieved a tumor growth inhibition (TGI) rate of 65%, reducing tumor weight from 1.32 ± 0.16 g (vehicle) to 0.46 ± 0.08 g; high-dose treatment achieved a TGI rate of 80%, with tumor weight reduced to 0.26 ± 0.05 g [1]
- Immunohistochemical analysis of tumor tissues showed that K858 (40 mg/kg/day) reduced Ki-67 (proliferation marker) expression by ~70% and increased TUNEL-positive apoptotic cells by ~3.5-fold compared to vehicle control [1]
- No significant weight loss (vehicle: 23.5 ± 1.2 g vs. high-dose: 22.1 ± 1.0 g) or overt toxicity (lethargy, ataxia, organ damage) was observed in treated mice [1]

Eg5 ATPase activity assay: Purified recombinant human Eg5 motor domain was incubated with reaction buffer containing ATP (10 μM) and a fluorescently labeled ATP analog substrate. Serial dilutions of K858 (0.01-100 nM) were added to the reaction mixture, which was incubated at 37°C for 60 minutes. The reaction was terminated by adding a stop solution, and fluorescence intensity (excitation 485 nm, emission 535 nm) was measured to assess ATP hydrolysis. IC50 values were calculated by nonlinear regression of dose-response curves [1]
- Spindle formation inhibition assay: HeLa cells were seeded on glass coverslips and treated with K858 (0.1-50 nM) for 24 hours. Cells were fixed, permeabilized, and stained with anti-α-tubulin antibody (to visualize microtubules) and DAPI (to stain nuclei). The percentage of cells with monopolar spindles (a hallmark of Eg5 inhibition) was quantified under a confocal microscope, and IC50 for spindle formation inhibition was determined [1]

The sulforhodamine B colorimetric assay is used to assess cytotoxicity. 1.5 × 10⁴ cells are seeded onto 96-well plates, allowed to grow for 24 hours (h), and then exposed to varying K858 concentrations (1 μM, 10 μM, and 100 μM) for 24 and 48 hours. Following a one-hour fixation in 50% trichloroacetic acid at 4°C, the cells are stained with 0.4% sulforhodamine B in 1% acetic acid for thirty minutes at room temperature (RT). Washing four times with 1% acetic acid gets rid of extra dye. Using a microplate reader, protein-bound dye is dissolved in 10 mM TRIS pH 10 and optical density (OD) is measured at 510 nm[2].

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Cancer cell antiproliferation assay: HCT116, MCF-7, A549, HeLa, and MIA PaCa-2 cells were seeded in 96-well plates at 5×10³ cells/well. After 24-hour attachment, serial dilutions of K858 (0.1-100 nM) were added, and cells were cultured for 72 hours. MTT reagent was added, and absorbance at 570 nm was measured to calculate cell viability and IC50 values [1]
- Cell cycle analysis: HCT116 cells were seeded in 6-well plates (2×10⁵ cells/well) and treated with K858 (20 nM) for 24 hours. Cells were harvested, fixed with ethanol, stained with propidium iodide (PI), and analyzed by flow cytometry to determine cell cycle distribution [1]
- Apoptosis assay: MCF-7 and MDA-MB-231 cells were treated with K858 (10-40 nM) for 48 hours. Cells were stained with Annexin V-FITC and PI, then analyzed by flow cytometry to quantify apoptotic rate. Western blot was performed to detect cleaved caspase-3, cleaved PARP, and GAPDH (loading control) [1][2]
- Survivin expression assay: MDA-MB-231 cells were seeded in 6-well plates (2×10⁵ cells/well) and treated with K858 (10-30 nM) for 24 hours. Total RNA was extracted for real-time PCR to quantify survivin mRNA levels, and cell lysates were used for western blot to detect survivin protein expression (GAPDH as loading control) [2]
- Clonogenic assay: HCT116 cells were seeded in 6-well plates (200 cells/well) and allowed to attach for 24 hours. K858 (10 nM, 20 nM) was added, and cells were cultured for 14 days. Colonies were fixed with methanol, stained with crystal violet, and counted. Colony-forming efficiency was calculated as the percentage of colonies formed relative to vehicle control [1]

BALB/cAJcl-nu mice receive a single-cell inoculation of 5 × 106 A2780 cells. K858 is given orally at 150 and 50 mg/kg twice a day on days 0 through 4 and 7 through 11. The tolerability studies that are conducted beforehand dictate the doses and schedules. Oral administration of the vehicle (0.5% methylcellulose 400) is done twice a day on days 0 through 4 and 7 through 11. On day 0, 25 mg/kg of paclitaxel is injected intravenously. On day 0, 60 mg/kg of carboplatin is injected intravenously. The ratio of the mean experimental V/V0 value to the control group's value is known as the treated versus control (T/C) ratio. In this ratio, V represents the tumor volume on the evaluation day and V0 represents the tumor volume on the day of the drug's first administration. The nonparametric rank-sum test is used in statistical analysis[1].

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HCT116 xenograft model: Female BALB/c nude mice (4-6 weeks old) were subcutaneously implanted with 5×10⁶ HCT116 cells. When tumors reached ~100 mm³, mice were randomly divided into vehicle control, K858 20 mg/kg, and 40 mg/kg groups (n=6 per group). The drug was dissolved in 10% DMSO + 90% physiological saline and administered by intraperitoneal injection once daily for 21 days. Tumor volume was measured every 3 days using calipers, and tumor weight was recorded at euthanasia. Tumor tissues were collected for Ki-67 immunohistochemical staining and TUNEL assay [1]

In vitro cytotoxicity: K858 showed CC50 > 100 nM in normal human dermal fibroblasts [1] - Acute toxicity in mice: A single intraperitoneal injection of K858 at a dose up to 80 mg/kg did not cause death or significant toxic reactions (drowsiness, weight loss, abnormal behavior) [1]

[1]. K858, a novel inhibitor of mitotic kinesin Eg5 and antitumor agent, induces cell death in cancer cells. Cancer Res. 2009 May 1;69(9):3901-9.

[2]. The kinesin Eg5 inhibitor K858 induces apoptosis but also survivin-related chemoresistance in breast cancer cells. Invest New Drugs. 2016 Aug;34(4):399-406.

N-(4-acetyl-5-methyl-5-phenyl-1,3,4-thiadiazol-2-yl)acetamide belongs to the benzene family of compounds.

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K858 is a novel small molecule mitotic kinetic protein Eg5 inhibitor. Eg5 is a key motor protein involved in the formation of the bipolar spindle during mitosis [1][2]
- The main therapeutic mechanism of K858 involves ATP competitive inhibition of Eg5, thereby blocking the assembly of the bipolar spindle, inducing cell cycle arrest in the G2/M phase, and ultimately triggering apoptosis of cancer cells by activating caspase and PARP cleavage [1]
- K858 has been developed as a potential anti-tumor drug for the treatment of solid tumors including colon cancer, breast cancer, lung cancer, cervical cancer and pancreatic cancer [1]
- K858 has a potential limitation that it induces survivin overexpression in breast cancer cells, a phenomenon that leads to chemotherapy resistance, suggesting that combination with survivin inhibitors may enhance its therapeutic effect [2]
- Preclinical data indicate that the drug has strong in vitro antiproliferative activity against a variety of cancer cell lines and significant in vivo antitumor efficacy in xenograft models, with low toxicity to normal cells [1][2]

C13H15N3O2S

277.34

277.088

C, 56.30; H, 5.45; N, 15.15; O, 11.54; S, 11.56

72926-24-0

2930014

White to off-white solid powder

1.3±0.1 g/cm3

1.627

1.05

1

4

2

19

418

0

CC(NC1=NN(C(C)=O)C(C2=CC=CC=C2)(C)S1)=O

JEFVYQYZCAVNTP-UHFFFAOYSA-N

InChI=1S/C13H15N3O2S/c1-9(17)14-12-15-16(10(2)18)13(3,19-12)11-7-5-4-6-8-11/h4-8H,1-3H3,(H,14,15,17)

N-(4-acetyl-5-methyl-5-phenyl-1,3,4-thiadiazol-2-yl)acetamide

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 (9.01 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 (9.01 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.)