Computational protocol: Selective Cytotoxicity of Amidinopiperidine Based Compounds Towards Burkitt’s Lymphoma Cells Involves Proteasome Inhibition

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Protocol publication

[…] Ramos cells (5×105 cells) were treated with the compound of interest for 24 h. The cells were then washed with PBS and subjected to fixation with 80% ethanol at –20°C for 15 min. Fixated cells were pelleted by centrifugation and rehydrated in 5 mL of PBS for 15 min at room temperature. Collected cells were resuspended in 500 µL staining buffer (3 µM propidium iodide, 100 mM Tris, pH 7.4, 150 mM NaCl, 1 mM CaCl2, 0.5 mM MgCl2, 0.1% Nonident P-40). After 15 min of incubation, samples were analysed on a FACScalibur and evaluated using FlowJo software (BD bioscience, Franklin Lakes/NJ, USA). [...] Km was determined by mixing 1 µg/mL of the purified human 20S proteasome (Boston Biochem, Inc., Cambridge/MA, USA) with various concentrations of the corresponding substrate ranging from 5 µM to 600 µM. Suc-Leu-Leu-Val-Tyr-AMC, Bz-Val-Gly-Arg-AMC and Z-Leu-Leu-Glu-AMC (Bachem, Bubendorf, Switzerland) were used for the chymotrypsin-like, trypsin-like and caspase-like activites, respectively. Assay buffer consisted of TE buffer (20 mM Tris (pH = 8.0), 0.5 mM EDTA, 0.03% SDS). Substrate hydrolysis was continuously monitored at 37°C with an automated microplate reader (Tecan Safire2). Measured fluorescence values (RFU) were plotted versus time with nonlinear regression to fit a Michaelis-Menten plot.For the Ki determination, 25 µL of substrate at three concentrations and 25 µL of inhibitor of interest also at three different concentrations were added to the wells of a white microplate. The reaction was initiated with 50 µL of the enzyme in the assay buffer. Flourescence of the AMC release was continuously monitored at 420 nm with 320 nm excitation.Km and Ki were calculated using SigmaPlot 11 (Enzyme Kinetics Module 1.3) and are presented as the mean of three independent experiments. [...] The computational study was carried out on a workstation with 4 dual-core AMD Opteron processors, 16 GB of RAM, a GeForce 7800 graphics card, and 1.2 TB of hard disk space, running the 64-bit Fedora 7 version. For the docking simulation, LeadIT 1.3.0 (BioSolveIT GmbH) was used on the crystal structure of the yeast 20S proteasome in complex with bortezomib (PDB entry: 2F16) , . Based on the kinetic studies the active site was defined as the area within 20Å of the N-terminal Thr1 residue of β2-subunit. Protonations and OH group orientations of the active-site amino acid residues were manually assigned with LeadIT GUI. For base placement Triangle Matching was used and the program generated maximally 200 solutions per iteration and 200 per fragmentation. We validated the system by the re-docking of co-crystallized bortezomib. For figure preparation the PyMOL Molecular Graphics System Version 1.3 was used (Schrödinger, LLC). […]

Pipeline specifications

Software tools SigmaPlot, LeadIt, PyMOL
Applications Drug design, Miscellaneous
Organisms Homo sapiens
Diseases Burkitt Lymphoma, Neoplasms, Drug-Related Side Effects and Adverse Reactions
Chemicals Benzamidines, Piperidines