Microtubule/Tubulin (colchicine binding site)

Bovine brain tubulin (0.4 mg) (Cytoskeleton, Denver, CO) was mixed with various concentrations (0.625-20 μM) of test compound and incubated in 120 μL of general tubulin buffer (80 mM PIPES, 2.0 mM MgCl2, 0.5 mM EGTA, pH 6.9, and 1 mM GTP). The absorbance of wavelength at 340 nm was monitored every 60 s for 20 min by the SYNERGY 4 microplate reader (Bio-Tek Instruments, Winooski, VT). The spectrophotometer was set at 37 °C for tubulin polymerization. The IC50 value was defined as the concentration which can inhibit 50% of microtubule polymerization.

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Cell Assay: Cell Culture and Cytotoxicity Assay of Melanoma. We examined the antiproliferative activity of the ATCAA and SMART analogues in one human melanoma cell line (A375) and one mouse melanoma cell line (B16-F1). We used activity on fibroblast cells as a control to determine the selectivity of these compounds against melanoma. A375 cells and B16-F1 cells were purchased from ATCC (American Type Culture Collection, Manassas, VA). Human dermal fibroblast cells were purchased from Cascade Biologics, Inc., Portland, OR. All cell lines were cultured in DMEM (Cellgro Mediatech, Inc., Herndon, VA), supplemented with 5% FBS (Cellgro Mediatech), 1% antibiotic/antimycotic mixture (Sigma-Aldrich, Inc., St. Louis, MO), and bovine insulin (5 μg/mL; Sigma-Aldrich). Cultures were maintained at 37 °C in a humidified atmosphere containing 5% CO2. Standard sulforhodamine B assay was used. Cells were exposed to a wide range of concentrations for 48 h in round-bottomed 96-well plates. Cells were fixed with 10% trichloroacetic acid and washed five times with water. After cells were air-dried overnight and stained with SRB solution, total proteins were measured at 560 nm with a plate reader. IC50 (i.e., concentration which inhibited cell growth by 50% of no treatment controls) values were obtained by nonlinear regression analysis with GraphPad Prism (GraphPad Software, San Diego,CA).

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SMART compounds bind potently to the colchicine binding site in tubulin, inhibit tubulin polymerization, and arrest cancer cells in the G2/M phase of the cell cycle. They equi-potently inhibit the growth of parental and MDR-overexpressing cells in vitro, indicating that they can overcome P-glycoprotein-mediated multidrug resistance.

In vivo anti-tumor efficacy studies in human prostate (PC-3) and melanoma (A375) cancer xenograft models demonstrated that SMART-H and SMART-F treatments resulted in %T/C values ranging from 4-30%. SMART compounds are similarly efficacious to currently FDA-approved antitubulin drugs for cancer treatment but can circumvent drug resistance.

A competitive mass spectrometry binding assay was developed to study the tubulin binding of SMART compounds, confirming that SMART-H specifically and reversibly binds only to the colchicine-binding site, and not to vinblastine- or paclitaxel-binding sites.

Antiproliferative activity was examined in A375 human melanoma and B16-F1 mouse melanoma cells using the SRB assay after 48 hours of compound exposure, with fibroblast cells used as a non-cancerous control. The compounds demonstrated potent cytotoxicity and broad-spectrum cellular activity.

In vivo anti-tumor efficacy was evaluated in human prostate (PC-3) and melanoma (A375) cancer xenograft models. SMART-H and SMART-F treatments resulted in tumor growth inhibition with %T/C values ranging from 4-30%. Treatment for 21 days at the higher dose (15 mg/kg) failed to produce any apparent neurotoxicity.

J Med Chem.2009 Mar 26;52(6):1701-11;J Med Chem.2011 Jul 14;54(13):4678-93.

C19H17NO4S

355.408

355.087

1135797-91-9

25227430

Typically exists as solid at room temperature

4.2

0

6

6

25

429

0

S1C=C(C(C2C=C(C(=C(C=2)OC)OC)OC)=O)N=C1C1C=CC=CC=1

WZEMDWPKLKAUOH-UHFFFAOYSA-N

InChI=1S/C19H17NO4S/c1-22-15-9-13(10-16(23-2)18(15)24-3)17(21)14-11-25-19(20-14)12-7-5-4-6-8-12/h4-11H,1-3H3

(2-phenyl-1,3-thiazol-4-yl)-(3,4,5-trimethoxyphenyl)methanone

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: soluble ( 10 mM)
Water: 1 mg/mL (Insoluble)
Ethanol: 1 mg/mL