In vitro activity: 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.

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).