Paclitaxel

Full-length myc-KIF1C synthesized in vitro with ³⁵S-methionine (EasyTag, Perkin Elmer, San Jose, CA) was desalted and incubated with GTPγS-preloaded Rabs (4.2 μM) for 1 hr in Buffer A (−BSA), 1 mM DTT, 2.5 mM ADP, 0.5 mM GTPγS. Microtubules (0.8 mg/ml), polymerized in 80 mM PIPES, 1 mM MgCl₂, 1 mM EGTA, pH 6.8 (BRB80), 1 mM DTT, 1 mM GTP, 10% DMSO, spun through a 40% glycerol cushion, and resuspended in BRB80, 1 mM DTT, 0.2 mM Paclitaxel (Cytoskeleton, Inc.), were incubated with the KIF1C-Rab complexes for 1 hr before being spun through a 10% sucrose, 20 µM Paclitaxel, 1 mM DTT, at 65K for 5 min (Optima TLX, Beckman Coulter, Inc., Indianapolis, IN). Scintillation counting and SDS-PAGE and radiography using a Typhoon imager (GE Healthcare Biosciences) revealed the amount of ³⁵S-labeled-KIF1C in fractions.

Common chemicals were purchased from Fisher Scientific and Sigma-Aldrich. Porcine brain tubulin, GTP and paclitaxel were purchased from Cytoskeleton, Inc. 99.8% D₂O, ¹⁵NH₄Cl and U-¹³C₆ glucose, 2-¹³C glucose, 1,6-¹³C glucose, U-¹³C₆,D₇ glucose were purchased from Cambridge Laboratories, Inc. EDTA-free protease inhibitor tablets were obtained from Roche. Chromatography columns were purchased from GE Healthcare. The 400 mesh copper grids coated with formvar and stabilized with evaporated carbon films were purchased from Electron Microscopy Science.
The human KIF5B motor domain construct (amino acids 1–349) was prepared using the pET28b vector fused with a His₆-SMT3 Tag at the N-terminus (in the form of His₆-SMT3-KIF5B). The His₆-SMT3-KIF5B was transformed into Escherichia coli BL21(DE3) cells. The His₆-Ulp1 was expressed and purified from the His₆-Ulp1 protease construct kindly provided by Dr. Christopher Lima from HHMI and Memorial Sloan Kettering Cancer Center, New York, NY 10065.

A customized drug library containing a total of 1,120 compounds was obtained from the Prestwick Chemical Library (Prestwick Chemical, Illkirch-Graffenstaden, France). All drugs in the drug library were solubilized in DMSO (Figure 1), subsequent experiments used other diluents (discussed below). The following chemicals were obtained from Sigma-Aldrich: fenbendazole (F5396), albendazole (A4673), fluspirilene (F100), clofazimine (C8895), niclosamide (N3510), suloctidil (S9384), tween-80 (P4780), N-methyl-2-pyrrolidone (NMP) (328634) and cremophor EL (Cr-EL) (C5135). Suloctidil and niclosamide were always prepared and used in DMSO (Figures 2, 4, 5). Fenbendazole, fluspirilene, and clofazimine were prepared in DMSO (Figures 1–3) or DNTC (Figures 3–5). Albendazole was prepared in DNTC or nanoparticles (PLGA) (Figure 6). Paclitaxel was obtained from Cytoskeleton (for in vitro) or Bristol-Myers Squibb (for in vivo). Gaussia luciferase system (LP-07) was purchased from Targeting Systems. Caspase inhibitor Z-VAD-FMK (03FK10901) and Matrigel (354323) were purchased from MP Biomedical and BD Biosciences, respectively.

The tubulin polymerization Assay (BK006P) was purchased from Cytoskeleton Inc. (Denver, CO, USA) and it was used with the purpose to measure the optical density of each sample over time using a spectrophotometer at a temperature of 37 °C at a wavelength of 355 nm on 96 well-plates. The tubulin protein was purified from porcine brain and diluted in General Tubulin Buffer (GTB), the final reaction concentration of tubulin was 3 mg/ml. Paclitaxel (Cytoskeleton Inc.) and colchicine (C9754, Sigma, St. Louis, MO, USA) were resuspended in DMSO and diluted in GTB for a final compound concentration of 10 µM. 25B-NBF (15967, Cayman Chemical, Ann Arbor, MI, USA), 25C-NBF (15969, Caymen Chemical, Ann Arbor, MI, USA), and DMPMPP (GLXC-22812, Glixx Labs, Hopkinton, MA, USA) were diluted in DMSO for a stock of 2 mM and then further diluted in GTB for different final concentrations of 10 µM, 50 µM, 75 µM and 100 µM. The final concentration of the polymerization reaction was 80 mM PIPES, 2.0 mM MgCl₂, 0.5 mM EGTA, 60% v/v glycerol, pH of 6.9, and 10 µM of guanosine triphosphate (GTP). Polymerization was measured every minute in a Victor Nivo (Perkin Elmer, Waltham, MA) at an optical density (OD) of 355 nm. The microplate was continuously disturbed using the orbital shaking function of the plate reader in between measurements as recommended by the manufacturer.