ed compounds is provided in the supplementary material. Computational Modelling PI3K X-ray structures were obtained from the PDB. All solvents and small molecules were removed from structures. Protein preparation and refinement was performed using Maestro 9.0 or 9.1 Protein Preparation Wizard default parameters were used to optimize protein structures. Cilomilast SB-207499 Receptor grid generation was confined to 20 Ǻ from the binding site ligand. Alignment of X-ray structures 2a5u and 2rd0 in Maestro was performed to determine the 2rd0 binding site. Ligands were constructed in Sybyl-X, energy minimized using the Tripos force field default settings for 1000 steps, imported into Maestro and prepared using LigPrep 2.3. Adjustment in protonation state was performed manually in Maestro. Docking calculations were performed in Glide 5.
5 or 5.6 using extra precision mode only. Sampling was limited to 10000 ligand poses per docking run and only one pose per ligand was retained. CI-1033 The set of 1000 drug-like decoy compounds of average molecular weight 360 was obtained from Schrödinger. The decoy set enriched with our seventy-three compounds was docked into each X-ray structure and ranked by GlideScore. Homology models of PI3Kα were built using the PI3Kδ crystal structure as the template. The structure was edited to 378 amino acids encompassing the catalytic domain only. Human PI3Kα and mus musculus PI3Kδ sequences were obtained from the National Center for Biotechnology Information and aligned using Protein BLAST. Homology models were generated in Prime with selected loops refined using extended sampling, then minimised.
Induced Fit docking utilising Prime and Glide XP mode was performed using default settings unless otherwise specified. Serono compound AS-605240 was docked initially for identification of optimal model protein structures. Receptor grid generation for each of the nine selected structures were prepared as described above. Docking calculations using XP mode for the nine structures using the Schrödinger decoy set enriched with our seventy-three compounds and ranked by GlideScore. ROC curves were generated using Microsoft Excel. Images were created using PyMOL. PI3Kα models 3 and 5 in pdb format with sets of 73 docked ligands for each of these models in sdf format are provided as supplementary material. Inhibition Assay PI3K protein was purified from cell lysates of transfected-Sf9 insect cells and diluted in assay buffer containing 2.
5 ug each of L-a-Phosphatidylinositol and 1,2-Diacyl-snglycero-3-phospho-L-serine in 96 well plates. Inhibitors were dissolved and diluted in DMSO. Reactions were started upon addition of 10 uM ATP with 40 μCi/ml γ-32P-ATP and 2 mM MgCl2. Reactions were incubated for 2 hours at room temperature, terminated upon addition of 2N HCl. Lipids were extracted in CHCl3/MeOH. Extracted organic fraction containing 32P-PI P was quantitated via addition of Microscint C using a TopCount 96 well plate scintillation counter measuring cpm. Prism 5 sold by GraphPad Software was used to calculate IC50 concentrations and inhibition curves. Pinson et al. Page 8 ChemMedChem. Author manuscript; available in PMC 2011 October 5.
HHMI Author Manuscript HHMI Author Manuscript HHMI Author Manuscript Supplementary Material Refer to Web version on PubMed Central for supplementary material. Acknowledgments We would like to thank Dr David Manallack with his assistance in calculating pKa values. We would also like to thank the Victorian Partnership for Advanced Computing and the Victorian Life Sciences Computation Initiative which are supported by the Australian Commonwealth and Victorian Governments. This work was funded though g
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