Computational protocol: Dual protein kinase and nucleoside kinase modulators for rationally designed polypharmacology

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[…] Raw files generated from mass spectrometry analysis were processed with Proteome Discoverer 1.3 (Thermo fisher Scientific). This software was used to search data via in-house Mascot server (version 2.4.1; Matrix Science Inc.) against the Human subset (20,264 sequences) of the SwissProt database (version 2014-03). For the database search the following settings were used: a maximum of one miss-cleavage, oxidation as a variable modification of methionine, carbamido-methylation as a fixed modification of cysteine and trypsin was set as the enzyme. A peptide mass tolerance of 6 p.p.m. and a fragment mass tolerance of 0.8 Da were allowed. Only peptides with high stringency Mascot Score were used for protein identification. Peptides FDR <1% was used. Each pull-down experiment was performed using two types of beads to select specific drug interactors from experimental contaminants: beads coupled to the drug and control beads without the drug. A complete list of identified proteins was enclosed in Supplementary Datas −.To go further in the analysis, relative intensity-based label-free quantification was processed using Progenesis LC−MS software (Progenesis QI for Proteomics v1.0; Nonlinear Dynamics) according to manufacturer instruction (Supplementary Datas , ). First, raw LC Orbitrap MS data were imported and LC−MS heatmap of retention time and m/z were generated. Features from these LC−MS were automatically aligned and filtered to retain signals crossing the following parameters; retention time 15–135 min, m/z 300–1700, charge state 2–6 and 3 or more isotopes. MS−MS spectra were exported into peak list as Mascot generic files (MGF) to allow protein identification using the following inclusion options; the 3 highest intensity precursors for each features and precursor intensity over 25%. MGF files were used to search data via in-house Mascot server (version 2.4.1; Matrix Science Inc.) against the Human database subset of the SwissProt database (version 2014.03). Database search were done using the following settings: a maximum of one trypsin miss-cleavage allowed, methionine oxidation and cysteine carbamido-methylation as fixed modification. A peptide mass tolerance of 6 p.p.m. and a fragment mass tolerance of 0.8 Da were allowed for search analysis. Only peptides adjusted to 5% false discovery rate and with ion score cutoff of 20 were exported from Mascot results and imported back to Progenesis LC−MS for protein grouping and quantification. The raw abundances of all identified peptides were used to normalise LC−MS−MS intensities. Total ion intensity signal from each of the individual peptides generated protein quantification. Any conflicting peptide identifications were removed from the measurements of the quantified proteins. Univariate one-way analyses of variance (ANOVA) were performed within Progenesis LC−MS to calculate the protein p-value according to the sum of the normalised abundances across all runs. [...] Sensitivity to gemcitabine and combination treatment with gemcitabine and masitinib were investigated by proliferation assays in cell lines using the CellTiter-Blue Reagent (Promega). The cells were plated in 96-well plates (5000 cells per well) in supplemented RPMI-1640 and/or DMEM media and exposed to either an increasing concentration of gemcitabine (0−100 µM) or masitinib (0−10 µM) or the combination of both and incubated for 5 days. At the end of incubation, 10 µl of CellTiter-Blue Reagent were added to each well. The absorbance was read at 590 nm. The effect of the drugs on cells was expressed as a percentage of proliferation compared to untreated cells. The experiments were performed in triplicates and data are presented as the mean ± s.d. Drug synergy effects were analysed using Combenefit (Cancer Research Cambridge Institute), a software tool that enables the visualisation, analysis and quantification of drug combination effects. The data from combination treatments were processed using three synergy reference models: highest single agent (HSA) model, Loewe additivity model and Bliss independence model for HRT18 and LnCAP cell lines. [...] Prior to data collection, crystals were rapidly soaked in a cryo-buffer, containing reservoir solution complemented with 25% (v/v) glycerol, and subsequently flash frozen in liquid nitrogen. X-ray data were collected at the European Synchrotron Radiation Facility (Grenoble, FRANCE) on beamline ID23-1 and ID23-2. The data reduction and scaling were performed using XDS suite. [...] The structure of dCK-C3S was solved by molecular replacement using PHASER and the chain A of the recently published dCK-C4S structure (PDB ID: 1P60) was used as starting model. The initial molecular replacement solution was further refined using BUSTER (Bricogne G., Blanc E., Brandl M., Flensburg C., Keller P., Paciorek W., Roversi P., Sharff A., Smart O.S., Vonrhein C., Womack T.O. (2011). BUSTER version 2.10.2. Cambridge, United Kingdom: Global Phasing Ltd.), alternating with cycles of manual rebuilding in COOT. Full data and refinement statistics are shown in Supplementary Table . […]

Pipeline specifications

Software tools Combenefit, XDS, Coot
Applications Drug design, Small-angle scattering, Protein structure analysis
Chemicals Deoxycytidine, Nucleosides