Crystal Structure of the PIM2 Kinase in Complex with a Ruthenium Half-Sandwich Inhibitor - Bullock et al.

Material and Methods

Protein expression and purification: Full length human PIM2 (34 kD isoform, gi 42821112) was subcloned by ligation-independent cloning into a pET-derived expression vector, pLIC, and expression performed in BL21(DE3) with 0.15 mM isopropyl 1-thio-β-D-galactopyranoside induction for 4 h at 18°C. Cells were lysed using a high pressure homogenizer and cleared by centrifugation, and the lysate was purified by nickel-sepharose chromatography. The eluted PIM2 protein was treated with λ-phosphatase together with tobacco etch virus (TEV) protease overnight to remove phosphorylation and the hexahistidine tag, respectively. The protein was further purified on a Mono Q column and by size exclusion chromatography. The eluted protein was homogeneous and non-phosphorylated as shown by ESI-MS. PIM2 protein was stored at 4°C in elution buffer (50 mM HEPES, pH 7.5, 250 mM NaCl) with 10 mM DTT or frozen in liquid nitrogen and stored at -80°C. Typical crystals had dimensions of 15 x 5 x 5 µm³.

Crystallization and Structure Determination: PIM2 was concentrated to 11 mg/ml in the presence of compound 1 which was added to an initial concentration of 0.6 mM (from a 10 mM stock solution in DMSO). Crystals were grown at 4°C in 1.5 µl sitting drops mixing 0.3 µl PIM2 with 1.2 µl mother liquor (90 mM HEPES pH 7.5, 1.44 M Na/KPO₄) and cryo-protected in mother liquor containing 30% glycerol.

PIM2 diffraction data were collected on a flash-cooled crystal (100 K) at the Swiss Light Source beamline SLS X10SA. Images were indexed and integrated using MOSFLM, and scaled using SCALA implemented in the CCP4 suite of programs. The structure was solved by molecular replacement using the program Phaser with the coordinates of PIM1 in complex with BIM1 (Protein Data Bank (PDB) code 1XWS). REFMAC5 was used for refinement with iterative rounds of rigid-body refinement and restrained refinement with TLS, against maximum likelihood targets, interspersed with manual rebuilding of the model using Xfit/XtalView.

Coordinates for the PIM2-inhibitor complex have been deposited in the Protein Data Bank (PDB code 2IWI).

Measurement of Protein Kinase Inhibition: The synthesis of all compounds has been reported recently [26,27]. PIM kinases (human) and substrate were purchased from Upstate Biotechnology USA. 10 nM concentrations of staurosporine or compounds 1-15 were incubated at room temperature in 20 mM MOPS, 30 mM MgCl₂, 0.8 µg/µL BSA, 5% DMSO (resulting from the inhibitor stock solution), pH 7.0, in the presence of substrate (50 µM S6 kinase/Rsk2 substrate peptide 2) and kinase (3.3 nM PIM1, 1.5 nM PIM2). After 15 minutes, the reaction was initiated by adding ATP to a final concentration of 100 mM, including approximately 0.2 µCi/µL [γ-³²P]ATP. Reactions were performed in a total volume of 25 µL. After 45 minutes, the reaction was terminated by spotting 17.5 µL on a circular P81 phosphocellulose paper (2.1 cm diameter, Whatman), followed by washing four times (5 minutes each wash) with 0.75% phosphoric acid and once with acetone. The dried P81 papers were transferred to a scintillation vial, and 5 mL of scintillation cocktail was added, and the counts per minute (CPM) were determined with a Beckmann 6000 scintillation counter. Each compound was measured in duplicate. Percent activity was calculated by dividing the averaged CPM for each compound by the control sample, corrected by the background.

Note: For further details about this kinase strutcure, please refer to SGC Material and Methods entry for PIM2.

References

26. Bregman H, Williams DS, Atilla GE, Carroll PJ, Meggers E (2004) An organometallic inhibitor for glycogen synthase kinase 3. J Am Chem Soc 126: 13594-13595.

27. Pagano N, Maksimoska J, Bregman H, Williams DS, Webster RD, et al. (2007) Ruthenium half-sandwich complexes as protein kinase inhibitors: derivatization of the pyridocarbazole pharmacophore ligand. Org Biomol Chem 5: 1218-1227.