Computational protocol: A π-Halogen Bond of Dibenzofuranones with the Gatekeeper Phe113 in Human Protein Kinase CK2 Leads to Potent Tight Binding Inhibitors

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Protocol publication

[…] Each of the three inhibitors 4b, 4a and 5 were separately crystallized with hsCK2α1–335. Prior to the crystallization these inhibitors were solubilized in 100% DMSO in a concentration of 10 mM. 4a and 5 were mixed with hsCK2α1–335 (8–10 mg/mL in 500 mM sodium chloride, 25 mM Tris/HCl pH 8.5) in a ratio of 1:10. 4b was mixed in a ratio of 1:5. After a short time of incubation, these mixtures were merged with reservoir solution (32% (w/v) PEG4000, 0.2 M ammonium acetate, 0.1 M citrate pH 5.6) in a ratio of 2.5:1. 3.5 µL of these mixtures were then equilibrated against the reservoir solution (4b and 4a: 1 mL; 5: 100 µL). The crystal growth was induced by seeding with 150 nL seeding solution after an equilibration time of two days. Grown crystals were harvested after one week.The crystals obtained in this way were cryo-protected by incubation in a vitrification solution [32% (w/v) PEG4000, 0.2 M ammonium acetate, 5% (v/v) glycerol, 0.1 M citrate pH 5.6, 2 mM of the co crystallized inhibitor] for 1 min followed by vitrification in liquid nitrogen.The subsequent X-ray diffractometry experiments were performed at beamline ID23-2 of the European Synchrotron Radiation Facility (ESRF) in Grenoble, France, and at beamline X06DA (PXIII) of the Swiss Light Source (SLS) in Villigen, Switzerland. We collected high resolution data from each of the hsCK2α1–335/inhibitor crystals (); in the case of the hsCK2α1–335 complexes with 4b and 4a we afterwards used the same crystal, respectively, to collect a “soft” X-ray diffraction data set (wavelength 2 Å) in order to unambiguously validate the positions of the inhibitors’ Cl-substituents via their contribution to anomalous diffraction. PDB codes: 4a: 5N9N; 4b: 5N9L; 5: 5N9K.All X-ray diffraction data were integrated with XDS [] followed by POINTLESS [] for symmetry determination, AIMLESS [] for scaling and CTRUNCATE as well as some other programs of the CCP4 software suite [] for the final steps of data reduction to structure factor amplitudes.The structures were solved by molecular replacement with PHASER [] using the PDB file 2PVR [] as a template. For refinement we used PHENIX [], manual modelling was made with Coot []. The topology of the three inhibitors were constructed with the help of PRODRG []. The anomalous difference density maps for the hsCK2α1–335 complexes with 4b and 4a were generated with the corresponding map calculation routine of PHENIX []. […]

Pipeline specifications

Software tools XDS, CCP4, PHENIX, Coot, PRODRG
Applications Drug design, Small-angle scattering, Protein structure analysis
Organisms Homo sapiens
Chemicals Amino Acids