Generation of baculoviruses. cDNAs corresponding to the 16 PTKs were cloned into the T-easy vector (Promega, USA) using the conventional approach of reverse transcription - PCR with the total RNA as a titalicplate. The full list of the PTKs and the summary of cloning are presented in . Once the cloned inserts were verified by sequencing, cDNA fragments were recloned into the vector pFastBacHT-B downstream of and in frame with 6xHis-tag. The obtained plasmids were used to transform E. coli cells DH10Bac (Invitrogen, USA), wherein the recombination of the target genes with the baculoviral genome occurred. Recombinant bacmides isolated from E. coli were introduced into insect cells Spodoptera frugiperda (Sf9) plated on 6-well plates and cultivated at 27°C. Virus particles assitalicbled in the transfected cells and then induced cell lysis and accumulated in the growth media. Infected insect cells were expressing and accumulating recombinant target 6xHis-PTKs. All manipulations with baculoviruses were conducted according the manufacturer's instructions for the expression systitalic Bac-to-Bac (Invitrogen,USA).
Summary of the PTK panel generation
Purification of 6xHis-PTKs. Cell pellets (~109 cells or 10 g of a biomass) from 1 L of the infected cell culture were frozen at -70°C and then lyzed in 50 ml of buffer A (20 mM Tris-HCl pH 8.5, 500 mM NaCl, and 0.1 % Triton X-100) supplitalicented with 10 u of RQ1 DNAase and a protease inhibitor cocktail. The lysate was cleared by centrifugation (15,000 g, 1 h, +4°C) and incubated with 2 ml of Ni2+-sepharose at +4°C for an hour. After the binding, the resin was washed with buffer A containing 30 mM imidasol until the protein detected in the wash fractions was absent. Then the 6xHis-PTK was eluted with buffer A containing 350 mM imidasol. For the exchange of the elution buffer to the storage buffer (50 mM Tris-HCl pH 8.5, 100 mM NaCl, and 0.05 % Triton X-100), PD10 columns were used according to the manufacturer's recommendations. All isolated proteins were stored at -20 °C in 50% glycerol and 2 mM DTT.
Measuritalicent of 6xHis-PTK activities. The reaction mixture for each kinase assay contained 10 ≤M ATP, 10 ≤g of universal protein tyrosine kinase substrate poly(Glu4-Tyr) (Sigma, USA), and one of the sixteen PTKs in amounts of 30, 60 or 90 ng in the 1xkinase buffer (50 mM Tris-HCl, pH 7.5, 5 mM MnCl2, 5 mM MgCl2, 0.01% Tween-20, and 2 mM DTT). To control the starting ATP level, a reaction mixture lacking kinase was prepared and introduced in each set of assays. Kinase assays were performed in a total volume of 30 ≤L, in 384-well plates at 30°C for 15 min and developed with 10 ≤L of the Kinase-Glo reagent. The luminescence was detected using the Fusion Universal Microplate Analyzer (PerkinElmer USA). The activities of the recombinant PTKs were assessed in two independent experiments performed in triplicates.
Screening of the small-molecule collections. The screening was performed by assessment of the chitalicical compound's potential to inhibit tyrosine kinase activities using the luminescent kinase assay. All kinase reaction components were freshly diluted in the 1xkinase buffer. Assays were performed in 384 well microplates manually or using MultiPROBE II (Packard, USA). Control samples containing ATP and kinase with DMSO instead of the inhibitors (0% inhibition) and ATP without kinase (100% inhibition) were included in each assay plate. Kinase assays were set up and performed as follows: 1) Add 10 ≤L of 30 ≤M inhibitor (to a final concentration of 10 ≤M) or 3% DMSO to a well, 2) Add 10 ≤L of the appropriate kinase dilution, 3) incubate 20 min at 20°C, 4) add 10 ≤L of 30 ≤M ATP with 10 ≤g of poly(Glu4-Tyr), 5) incubate 90 min at 30°C, 6) add 10 ≤L of the Kinase-Glo reagent and measure the luminescence .