Eg5 (kinesin spindle protein).

In the Eg5 motor domain, S-Trityl-L-cysteine binds to a specific pocket made up of multi-layered structural elements (helix a2/loop L5/helix a3) [1]. Through a pro-cue-mediated mechanism, S-Trityl-L-cysteine (1–20 μM) causes cell contraction and cell cycle arrest for 72 hours. The activation of mitogen-activated phospholipids and nuclear factors results in the inhibition of S-tritonyl-L-cysteine and cell cycle arrest. S-tritiuml-L-cysteine (IC50 = 700 nM) induces a mitotic signal in HeLa cells, which are characterized by a single star-shaped spindle [2].

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S-trityl-L-cysteine (STLC) treatment (0, 1, 5, 10, 20 μmol/L for 72 h) induced dose-dependent cell apoptosis in SH-SY5Y, SK-N-SH, and SK-N-BE2 neuroblastoma cells, with notable increase at 5 μmol/L compared to control [1].
Flow cytometric analysis showed that S-trityl-L-cysteine (STLC) induced cell cycle arrest at G2/M phase in a dose-dependent manner; the shift from G1 to G2/M phase was notable at 5 μmol/L [1].
Western blotting revealed that S-trityl-L-cysteine (STLC) treatment (48 and 72 h) decreased Eg5 protein expression in SY5Y cells in a dose-dependent manner [1].
S-trityl-L-cysteine (STLC) (1 or 5 μmol/L for 48 h) did not affect MYCN gene amplification in BE2 cells as detected by fluorescence in situ hybridization [1].
mRNA microarray analysis (5 μmol/L STLC for 72 h) demonstrated that S-trityl-L-cysteine (STLC) activates MAPK and NF-κB signaling pathways in SY5Y cells compared to control [1].

Cell lines (SH-SY5Y, SK-N-SH, SK-N-BE2) were maintained in DMEM supplemented with 10% fetal bovine serum at 37°C in 5% CO2 humidified atmosphere [1].
For apoptosis assay, cells were seeded into 12-well plates and treated with 0, 1, 5, 10, or 20 μmol/L S-trityl-L-cysteine (STLC) for 72 h. Cells were harvested, washed with PBS, stained with Annexin V and 7-AAD for 1 h at 37°C using an apoptosis detection kit, and analyzed by flow cytometry [1].
For cell cycle analysis, 3x10^4 cells were seeded into 12-well plates, treated with 1, 5, 10, or 20 μmol/L S-trityl-L-cysteine (STLC) for 72 h, harvested, fixed, stained with 10% propidium iodide for 2 h at 37°C, and analyzed by flow cytometry to determine percentage of cells in G1, S, and G2-M phases [1].
For immunofluorescence, cells plated on glass coverslips were fixed with 3% paraformaldehyde, permeabilized with 0.15% Triton X-100 for 12 h at 4°C, incubated with primary antibody (anti-Eg5, 1:200) for 1 h at room temperature, followed by Alexa Fluor 488 secondary antibody for 45 min, and nuclei stained with DAPI (1 μg/ml) for 20 min at room temperature [1].
For western blotting, cells were lysed in RIPA buffer with protease and phosphatase inhibitors. Protein concentration measured by BCA assay. 30 μg protein per sample separated by 15% SDS-PAGE, transferred to nitrocellulose membranes, blocked with 5% BSA, probed with anti-Eg5 (1:400) and anti-β-actin (1:5,000) overnight at 4°C, then HRP-conjugated secondary antibody for 1 h at room temperature, and scanned using LAS-4000 Mini system [1].
For FISH, BE2 cells treated with 1 or 5 μmol/L S-trityl-L-cysteine (STLC) for 48 h, harvested, washed. Vysis LSI N-MYC SO Probe was used. Probe mixture (1 μl probe, 2 μl ddH2O, 37 μl hybridization buffer) centrifuged, 10 μl added onto slide, coverslipped, heated at 72°C for 2 min, then incubated in humidified chamber at 37°C overnight. Slides were washed in 0.4X SSC at 75°C for 30 min, then 2X SSC/0.1% NP40 at 37°C for 1 min, air dried, stained with DAPI II for 1 h, and examined by epi-fluorescence microscopy at x1000 magnification [1].
For mRNA microarray, SY5Y cells treated with 0 or 5 μmol/L S-trityl-L-cysteine (STLC) for 72 h, total RNA isolated using TRIzol and purified with RNeasy Mini kit. RNA processed with GeneChip one-cycle target labeling kit, converted to double-stranded cDNA, biotinylated cRNA targets generated and hybridized to GeneChip PrimeView/U133 plus 2.0 Human Gene Expression Array. Arrays stained with Cy5-dCTP in Fluidics Station 450 at 45°C for 16 h and scanned on Affymetrix Scanner 3000. Data normalized with MAS 5.0/RMA algorithm using Gene Spring Software 11.0 [1].

[1]. S-trityl-L-cysteine, a novel Eg5 inhibitor, is a potent chemotherapeutic strategy in neuroblastoma. Oncol Lett. 2018 Jul;16(1):1023-1030.

[2]. In vitro screening for inhibitors of the human mitotic kinesin Eg5 with antimitotic and antitumor activities. Mol Cancer Ther. 2004 Sep;3(9):1079-90.

(2R)-2-amino-3-[(triphenylmethyl)thio]propionic acid is a benzene-based aromatic compound. Triphenylmethylcysteine is a derivative of cysteine and possesses antimitotic activity and potential antitumor activity. (NCI04)

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S-trityl-L-cysteine (STLC) is a selective allosteric inhibitor of Eg5, which blocks bipolar spindle formation causing mitotic arrest and apoptotic cell death. It has higher potency compared to monastrol or terpendole E in inducing mitotic arrest [1].
Eg5 is overexpressed in neuroblastoma tissue specimens and cell lines, with higher expression in BE2 cells (MYCN amplified) compared to SY5Y and SK cells. However, STLC antitumor activity is not dependent on MYCN amplification [1].
STLC treatment (5 μmol/L) significantly activates NF-κB and MAPK signaling pathways at the transcriptional level, which may contribute to apoptosis and cell cycle arrest [1].
This study is the first to demonstrate cell apoptosis and cell cycle arrest in neuroblastoma cell lines induced by S-trityl-L-cysteine (STLC) [1].

C22H21NO2S

363.4726

363.129

2799-07-7

76044

White to off-white solid powder

1.2±0.1 g/cm3

524.7±50.0 °C at 760 mmHg

182-183 °C (dec.)(lit.)

271.2±30.1 °C

0.0±1.4 mmHg at 25°C

1.642

5.56

2

4

7

26

392

1

C1=CC=C(C=C1)C(C2=CC=CC=C2)(C3=CC=CC=C3)SC[C@@H](C(=O)O)N

DLMYFMLKORXJPO-FQEVSTJZSA-N

InChI=1S/C22H21NO2S/c23-20(21(24)25)16-26-22(17-10-4-1-5-11-17,18-12-6-2-7-13-18)19-14-8-3-9-15-19/h1-15,20H,16,23H2,(H,24,25)/t20-/m0/s1

(2R)-2-amino-3-tritylsulfanylpropanoic acid

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 : ~25 mg/mL (~68.78 mM)

Solubility in Formulation 1: ≥ 1 mg/mL (2.75 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 10.0 mg/mL clear DMSO stock solution to 400 μL of PEG300 and mix evenly; then add 50 μL of Tween-80 to the above solution and mix evenly; then add 450 μL of 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: ≥ 1 mg/mL (2.75 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 10.0 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: ≥ 1 mg/mL (2.75 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 10.0 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.)