To explicit SRPK1 can increase DDP resistance of glioma cells in molecular levels

ates of all ligands were set at pH=7.4 using Epik. Docking–The virtual Sodium laureth sulfate screening was implemented in two consecutive steps using Schrodinger Glide Virtual Screening Workflow. Initially, the whole 3.7-million-compound library was docked into the receptor using Glide HTVS mode. The resulting top 100,000 ligands were retained for docking with Glide SP where six hydrogen bond constraints were set based on previous analysis.13: N of F223, N of K187, OE2 of E186, O of G163, O of water W1025, and O of D161. The top 777 compounds satisfying at least 4 out of the six constraints were retained and clustered based on their chemical similarity. Twelve representatives of each cluster and 12 SAM analogs occupying a previously identified hydrophobic cleft at Val249, including 5-ITC, were selected for experimental validation. 5.3. Differential scanning fluorimetry screening Thermal denaturation experiments were carried out in a Mx3005p real-time PCR machine using a protein concentration of 2 M and an inhibitor concentration of 10 M. Samples were buffered in 10 mM HEPES, pH 7.5, 500 mM NaCl and a 1:1000 dilution of Sypro Orange. The assay and data evaluation were carried out as previously described.20 Europe PMC Funders Author Manuscripts Europe PMC Funders Author Manuscripts Bioorg Med Chem. Author manuscript; available in PMC 2016 March 07. Yu et al. Page 7 5.4. DOT1L methyltransferase assay The assay conditions reported by Daigle et al were used with minor modifications.4 Nucleosome purified from chicken blood cells was used as substrate. Nucleosome was added into 20 l assay mixture with inhibitors at different concentrations or DMSO as a control). The mixture was incubated at room temperature for 30 minutes before 3H-SAM was added to start the reaction. Reaction mixture was incubated at room temperature for one hour and quenched by adding 160 l of 10% trichloroacetic acid. The mixture was transferred into a glass fiber filter plate and washed twice with 10% TCA and once with ethanol. 100 l Microscint Zero was finally added into filter plates and centrifuged to flow through filters. Tritium incorporation was monitored using TopCount. 5.5. Crystallization Purified protein DOT1L was concentrated to 16 mg/mL in a buffer containing 20 mM Tris-HCl pH 8.0, 200 mM NaCl, 1 mM EDTA, and 1 mM TCEP. To prepare the complex, PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19811088 DOT1L was mixed with BrSAH by directly adding a 5 fold excess of compound to the protein solution and crystallized by using the sitting drop vapor diffusion method at 18 C. Crystals of DOT1L with BrSAH were grown in conditions containing 1.5 M 2SO4, 100 mM sodium acetate, pH 4.6. Prior to flash-freezing the crystals in liquid nitrogen, the crystals were soaked in reservoir solution with added glycerol as a cryoprotectant. 5.6. Diffraction data collection and model refinement Europe PMC Funders Author Manuscripts Europe PMC Funders Author Manuscripts Diffraction data were collected at GM/CA collaborative access team beamline 23ID-D of the Advanced Photon Source, Argonne National Laboratory, at a wavelength of 1.0332. For the refinement data set, intensities from one hundred consecutive single-degree oscillation frames were integrated with DENZO and scaled with SCALEPACK.21 Amplitudes were derived with the program TRUNCATE.22 The unit cell was isomorphous to Protein Data Bank entry 3QOW8 which provided initial coordinates for refinement, as well as flags for cross-validation.23 Geometry restraints for BrSAH were calculated with PRODRG.24 The cu