Computational protocol: Optimized S-Trityl-l-cysteine-BasedInhibitors of Kinesin Spindle Proteinwith Potent in Vivo Antitumor Activity in Lung Cancer Xenograft Models

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Protocol publication

[…] Compounds 4 and 2 were purchased from Nova Biochem, Sigma Aldrich, and Selleck Chemicals, respectively, and used without further purification. Compound 1 was a gift from Sanofi-Aventis. Compounds 8, 9, 11–16, and 43 were prepared as reported previously. Compound 7 (NSC123528) was obtained from the NCI/DTP Open Chemical Repository ( of the National Cancer Institute. All reagents and solvents were of commercial quality and used without further purification. Anhydrous reactions were carried out in oven-dried glassware under a nitrogen atmosphere unless otherwise noted. Microwave reactions were performed using a Biotage Initiator-8 microwave synthesizer (operating at 2.45 GHz). Thin-layer chromatography (TLC) was carried out on aluminum-backed SiO2 plates (silica gel 60, F254), and spots were visualized using ultraviolet light (254 nm) or by staining with phosphomolybdic acid (alcohols) or ninhydrin (amines). Flash column chromatography was performed on silica gel (SNAP KP-Sil, 60 Å, 40–63 μm cartridges) using a Biotage SP4 automated chromatography system (detection wavelength, 254 nm; monitoring, 280 nm). Melting points were determined using a Stuart Scientific SMP1 melting point apparatus and are uncorrected. 1H and 13C NMR spectra were recorded on a JEOL ECX-400 (400 MHz), Avance DPX400 (400 MHz), or Avance DPX500 (500 MHz) spectrometer. 19F NMR spectra were recorded on an Avance AV400 (400 MHz) instrument equipped with a multinuclear probe. 1H chemical shifts (δ) are reported in ppm relative to the residual signal of the deuterated solvent (7.26 in CDCl3, 3.31 in CD3OD, and 2.50 in DMSO-d6). Multiplicities are indicated by s (singlet), d (doublet), t (triplet), q (quartet), m (unresolved multiplet), and br (broad signal). 13C chemical shifts (δ) are reported in ppm relative to the carbon resonance of the deuterated solvent (77.16 in CDCl3, 49.00 in CD3OD, and 39.52 in DMSO-d6). 19F spectra are referenced relative to CFCl3. High-resolution mass spectra were recorded on a Thermo Electron LTQ ORBITRAP mass spectrometer using electrospray ionization. Gas chromatography mass spectra (GC-MS) using electron ionization (EI) were recorded on a Thermo Scientific Focus GC with DSQ2 single quadrupole mass spectrometer. GC-MS using chemical ionization (CI) were recorded on an Agilent Technologies 7890A GC system and an Agilent 5975C Inert XL EI/CI MSD with a DSQ2 single quadrupole mass spectrometer, equipped with an Agilent Technologies DB5-MS column (30 m × 0.25 mm × 0.25 μm). Helium was the carrier gas (flow rate = 1 mL/min–1). Elemental analysis data were recorded on a Perkin-Elmer 2400 series 2 CHN analyzer. LC-MS analyzes were performed with an Agilent Quaternary 1200 series pump and an Agilent 6130 dual source mass spectrometer with UV detection at 254 nM. Retention times (tR) were in minutes, and purity was calculated as percentage of total area. The method for determining purity consisted of the following: Zorbax Eclipse XDB-C18 reverse phase column (15 cm × 4.3 μm, particle size 5 μm); column temperature 40 °C; solvent A: H2O (5 mM ammonium acetate); solvent B: MeCN (5 mM ammonium acetate); gradient of A:B, 95:5 (0–3 min), A:B, 95:5 → B, 100% (3–17 min), B, 100% (17–27 min), B, 100% → A:B, 95:5 (27–33 min), A:B, 95:5 (33–36 min); flow rate 1 mL min–1. All tested compounds were ≥95% pure by elemental or LCMS analyses. New compounds were named according to IUPAC nomenclature by ACD ChemSketch 12.01 (Windows, Advanced Chemistry Development, Toronto, Canada). [...] Cells were seeded in triplicate in 96-well assay plates at 1.250 cells (BxPC-3, HCT116), 1.500 cells (PC-3), 2.500 cells (NCI-H1299), 3.000 cells (LNCaP), or 5.000 cells (K562) per well in 100 μL of the respective growth medium. Medium blanks and cell blanks were also prepared for every cell line. On the next day, inhibitors were added with a starting concentration of 100 μM in a 3-fold serial dilution series. At 72 h after addition of inhibitors, 10% Alamar blue (Invitrogen, Paisley, U.K.) was added, and depending on the cell line, between 2 and 12 h later the absorbance was measured at 570 and 600 nm. All values were corrected for the absorbance of the medium blank, and the corrected cell blanks were set to 100%. Calculations for determining the relative proliferation were performed using the equations described in the manufacturer’s manual. The GI50 values were subsequently determined using a sigmoidal dose–response fitting (variable slope) with GraphPad Prism 5.03 for Windows (GraphPad Software, San Diego, CA). [...] Diffraction data for the Eg5–inhibitor complexes Eg5–46 were recorded at the ESRF ID23-2. Data were processed using iMosflm and scaled using Scala from the CCP4 suite of programs. The calculation of Rfree used 5% of data. The Eg5–46 structure was solved by molecular replacement (Phaser) using one molecule of Eg5 motor domain from 3KEN. Refinement was carried out with PHENIX. Electron-density and difference density maps, all σA-weighted, were inspected, and the model was improved using Coot. Model geometry was analyzed using MolProbity. For the Eg5–46 complex, 97.0% (322) of the residues are in the preferred regions, 2.7% (9) are in the allowed regions, and 0.3% (1) are outliers. Figures were prepared using PyMOL. […]

Pipeline specifications

Software tools Dr Fit, iMosflm, CCP4, PHENIX, Coot, MolProbity, PyMOL
Applications Drug design, Small-angle scattering, Protein structure analysis
Organisms Mus musculus, Homo sapiens
Diseases Lung Neoplasms, Neoplasms, Hematologic Neoplasms