Specific inhibition of CK2α from an anchor outside the active site

CAM4066, a specific CK2α kinase inhibitor, is anchored in the cryptic αD pocket outside the active site and inserts a “warhead” into the active site, blocking ATP binding and thereby inhibiting the kinase.

Electronic Supplementary Material (ESI) for Chemical Science. This journal is © The Royal Society of Chemistry 2016

Protein expression and purification
Four constructs of CK2α were used in this study. For ITC and kinase activity assays CK2α_WT was used (residues 2-329). For crystallization purposes three different constructs were used: CK2α_KA, CK2α_FP10 and CK2α_FP9. CK2α_KA (residues 2-329) contained four mutations designed to aid crystallization by reducing the overall charge of the protein; R21S, K74A, K75A and K76A. CK2α_FP10 contained one mutation (R21S) and an N-terminal extension GSMDIEFDDDADDDGSGSGSGSGS aimed at mimicking a substrate peptide for CK2α. CK2α_FP9 contained the same R21S mutation and the Nterminal extension GSMDIEFDDDADDDGSGSGSGSG. CK2α_FP10 and CK2α_FP9 were cloned into pHAT4 vector and CK2α_KA was cloned into pHAT2 vector to give constructs with cleavable His 6tags.
Recombinant plasmids containing one of the four constructs (CK2α_WT/ CK2α_KA/ CK2α_FP10/ CK2α_FP9) were introduced into Escherichia coli BL21 (DE3) for protein production. Single colonies of the cells were grown in 6x1 L of 2xTY with 100 μg/mL ampicillin at 37 °C. Isopropyl thio-β-Dgalactopyranoside (IPTG) was added to a final concentration of 0.4 mM to induce expression when the optical density at 600 nm reached 0.6. The cells were incubated overnight at 25 °C then harvested by centrifugation at 4,000 g for 20 minutes. The same extraction and purification procedure was used for all four constructs, with the exception that CK2α_KA used 350 mM NaCl in the buffer, whereas, CK2α_WT and CK2α_FP10/9 required 500 mM NaCl. The cell pellets were suspended in 20 mM Tris, 350/500 mM NaCl, pH 8.0) and lysed using a high pressure homogenizer. Protease inhibitor cocktail tablets (one tablet per 50 mL extract; Roche Diagnostics) and DNase I were then added. The crude cell extract was then centrifuged at 10,000 g for 45 minutes, the supernatant was filtered with a 0.22 μm filter. The soluble supernatant was applied on a Ni Sepharose Fast Flow6 column at pH 8.0, washed and eluted in 20 mM Tris pH 8.0, 350/500 mM NaCl, 200 mM imidazole. After overnight dialysis into 20 mM Tris, pH 8.0, 350/500 mM NaCl the Nterminal His 6 -tag was cleaved overnight by TEV protease and passed through a second metal affinity column to remove uncleaved protein and the protease. The cleaved protein was further purified on a Sepharose Q HP anion-exchange column and the main peak fraction from this column was further purified by gel filtration on a Superdex 75 16/60 HiPrep column equilibrated with Tris 20 mM, pH 8.0, 350/500 mM NaCl. Pure protein was concentrated to 15 mg/mL and flash frozen in liquid nitrogen.

X-ray crystallography
CK2α_KA at 5 mg/mL in 20 mM Tris, pH 8.0, 350 mM NaCl, 1 mM DTT, and 25 mM ATP was crystallised with 112.5 mM MES pH 6.5, 35% glycerol ethoxylate and 180 mM ammonium acetate in a 1:1 ratio with a total volume of 2 µL by the hanging drop vapour-diffusion method. The fragments were soaked as singletons at 2-100 mM into these crystals for 15-20 h in 107 mM MES pH 6.5, 35% glycerol ethoxylate and 1.04 M ammonium acetate after which the crystals were cryo-cooled in liquid nitrogen for data collection.
CK2α_FP10 at 10 mg/mL in 20 mM Tris, pH 8.0, 500 mM NaCl, 4 mM DTT, 13 mM ATP, 2 mM phytic acid was crystallised with 107 mM MES, pH 6.5, 29% glycerol ethoxylate, 1.04 M ammonium acetate in a 1:1 ratio with a total volume of 2 µL by the hanging drop vapour-diffusion method. The fragments were soaked into the crystals of CK2α_FP10 for 15-20 h at 100 mM in 107 mM MES pH 6.5, 29% glycerol ethoxylate and 1.04 M ammonium acetate. The crystals were cryo-cooled in liquid nitrogen in the same solution for data collection. CK2α_FP9 at 5 mg/mL in 20 mM Tris, pH 8.0, 500 mM NaCl, 4 mM DTT, 8 mM ATP was crystallised with 92 mM MES, pH 6.5, 33% glycerol ethoxylate, 750 mM ammonium acetate in a 1:1 ratio with a total volume of 2 µL by the hanging drop vapourdiffusion method. The crystals were cryo-cooled in liquid nitrogen in the same solution for data collection.
Fragment screen to identify ATP site fragment was performed using 348-fragment Zenobia "ZEN-LIBRARY 1" library. The fragments were soaked as cocktails of four at 5 mM concentration for each fragments into CK2α_KA crystals in 15-20 h in 107 mM MES pH 6.5, 35% glycerol ethoxylate and 1.04 M ammonium acetate after which the crystals were cryo-cooled in liquid nitrogen for data collection.
X-ray diffraction data was collected at ESRF and Diamond synchrotron radiation sources, then processed using the pipedream package by Global Phasing Ltd; structures were solved by using programs from the CCP4 package. 25 Models were iteratively refined and rebuilt by using AutoBuster 26 and Coot programs. Ligand coordinates and restraints were generated from their SMILES strings using the Grade 27 software package. F o -F c electron density maps for all the ligands, calculated prior to ligand placement in the model, are shown in Figure S4 and stereo figures of CAM4066 density in Figure S5. All coordinates have been deposited to Protein Data Bank and accession numbers and data collection and refinement statistics are shown in Table S3 with crystallisation and soaking conditions listed in Table S4.

ITC
All ITC experiments were performed at 25 °C using a MicroCal itc200 instrument (GE Healthcare). CK2α_WT (20 mg/mL, 20mM Tris pH 8.0, 500 mM NaCl) was diluted in Tris buffer (200 mM Tris, 300 mM NaCl, 10% DMSO) and concentrated to 20-50 μM. Compounds in 100x stock solutions were diluted into the buffer ensuring that the DMSO concentrations were carefully matched. In a typical experiment CK2α_WT (40 μM) was loaded into the sample cell and 0.4-2.0 mM of the ligand was titrated in nineteen 2 μL injections of 2 s duration at 150 s intervals, with injector speed of 750 rpm. Heats of dilution were determined in identical experiments, but without protein in the cell. The data fitting was performed with a single site binding model using the Origin software package.

Kinase assays
The kinase assays were performed using the ADP-Glo™ kinase assay kit (Promega). 50 nM CK2α_WT was incubated in the kinase reaction buffer (40 mM Tris pH7.5, 200 mM NaCl, 20 mM MgCl 2 , 0.1 mg/mL BSA, 25 μM ATP, 50 μM substrate peptide (RRRADDSDDDD, Enzo Life Sciences Inc.), 5% (v/v) DMSO) in the presence of different concentrations of the inhibitor at 25 °C for 40 min. 5 μL aliquots of the kinase reaction were quenched with 5 μL of ADP-glo™ solution. After another 40 min the kinase detection reagent was added and maintained at 25 °C for 30 minutes. The luminescence was recorded using a PHERAstar FS plate reader (BMG LABTECH) with an integration time of 1 s. Percentage inhibition was calculated relative to a DMSO control and a baseline measurement without ATP. All measurements were performed in triplicate. The IC 50 curves were fitted using Sigma plot 11.0.

Cell culture
All cell lines used were obtained from ATCC and were supplied as mycoplasma free. HCT116 colon carcinoma cells were maintained in McCoy's 5A (1x) + Glutamax-I growth medium (Gibco, 36600-021) supplemented with fetal bovine serum (FBS, Gibco Life Technologies, 10270-106) at a final concentration of 10 %. A549 lung adenocarcinoma cells were cultured in Dulbecco Modified Eagle medium (DMEM) (1x) +Glutamax-I (Gibco Life Technologies, 31966-021) with 10% FBS. Jurkat T-cell lymphoblastic cells were cultured in RPMI 1640 (Gibco Life Technologies, 72400-021) growth medium supplemented with penicillin-streptomycin (Sigma, P0781) and FBS with the final concentrations of each being 1% and 10% v/v respectively. All cells were grown at 37 °C/ 5 % CO 2 in a humidified environment and all the assays were performed using these culturing conditions.

Growth Inhibition assays
Adherent cell lines (HCT116 and A549 cells) were seeded into flat-bottomed tissue culture 96-well plates in a volume of 150 µL of growth medium. HCT116 cells were seeded at 750 cells per well and A549 cells were seeded at 1000 cells per well. After 24 hours, compounds dissolved in DMSO were diluted in growth medium and were added to cells such that the final DMSO concentration was 1% (v/v) and the final volume in the well was 200 µL. Cells were then incubated in the presence compound for 72 hours before fixation.
Medium was removed from cells and 100 µL of cold 1% (v/v) trichloroacetic acid was added and the plates were incubated for 30 minutes at 4 °C after which the acid was removed and the plates were washed three times in tap water and left to dry at room temperature. The fixed cells were stained in a 0.057% sulforhodamine B/1% acetic acid solution (w/v) and incubated at room temperature with agitation for 30 minutes after which the dye was removed and the plates washed in 1% (v/v) acetic acid and left to dry. The dye was then solubilised in 10 mM Tris solution (pH8) and incubated for 30 minutes under agitation. The plates were then read on a PHERAstar plus plate reader (BMG Labtech) using the fluorescence intensity module (540-590 nm). Growth inhibition was calculated relative to DMSO controls and GI 50 values were calculated using Graphpad Prism.
Jurkat cells were seeded (20,000 cells per well) in growth medium in a 96-well flat-bottomed plate and immediately dosed with compounds (dissolved in DMSO) such that the final volume in the well was 200 µL and 1% DMSO (v/v). Cells were incubated for a further 72 hours. After this time, 5% (v/v) of CellTitre-Blue reagent (Promega) was added to each well and incubated for a further 2 hours under normal tissue culture condition, described above. The fluorescence was then measured using the PHERAstar plus plate reader (BMG Labtech) using the fluorescence intensity module (540-590 nm). Growth inhibition was calculated relative to DMSO control and GI 50 values were calculated using Graphpad Prism.

Western Blotting
HCT116 cells (2 ml) were seeded into 6-well tissue culture plates at a seeding density of 3x10 5 cells/ml and cultured for 24 hours prior to the addition of compound. Compound was diluted in culture medium to the desired concentration and a final DMSO concentration of 1% (v/v). Cells were harvested by trypsination (growth medium was collected and included in the analysis), washed in PBS and the pellet collected. Cells were lysed using a NP-40 lysis buffer (50 mM Tris pH 8, 150 mM NaCl, 1% NP-40) with the addition of Proteoblock protease inhibitor (Fermentas), and the phosphatase inhibitors #2 and #3 (Sigma Aldrich) at the recommended concentrations. The cell pellet was incubated in lysis buffer on ice for 2 hours and then centrifuged for 10 minutes at 4 °C at 13000 rpm on a bench top centrifuge for 10 minutes and the supernatant collected and stored at -80 °C. Protein levels were quantified using the Pierce BCA protein assay kit (Pierce, Thermo Fisher Scientific).
A total of 30 µg of protein was loaded onto a 4-12% Bis-Tris gel (Invitrogen) and run for 1 hour at a constant 200 V. The gel was transferred onto PVDF membrane at 4 °C overnight at a constant 50 V. Transfer efficiency was confirmed by staining the membrane with Ponceau S (Sigma-Aldrich) after which it was incubated in blocking buffer (either 5 % Milk-TBS-0.1 % Tween 20 or 5 % BSA-TBS-0.1 % Tween 20) for 1 hour. Membranes were then incubated with either anti-AKT1 (phosphoS129) (Abcam, ab133458) or anti-Cdc37 (phospho S13) (Abcam, ab108360) antibodies diluted in 5% BSA-TBST for 24 hours at 4 °C. Anti-β actin was used as a loading control (Sigma-Aldrich, A5441) diluted in Milk-TBS-0.1% Tween 20. After washing, membranes were then incubated in HRP-labelled antirabbit antibody for 1 hour at room temperature and then visualised using ECL (GE Healthcare). Where membranes were stripped for re-probing, membranes were immersed in Restore Western Blot stripping buffer (Thermo Scientific) for 15 minutes at room temperature.
The ammonium salt S3 was prepared as described in scheme S3.

General experimental details:
Solvents: Except as otherwise indicated, reactions were carried out using oven-dried glassware under nitrogen with dry, freshly distilled solvents. THF was distilled from CaH 2 and LiAlH 4 in the presence of triphenylmethane. Diethyl ether was distilled from CaH 2 and LiAlH 4 . CH 2 Cl 2 and MeOH were distilled from CaH 2 . All other solvents were used as obtained from commercial sources.  Melting points: These data were collected on a BÜCHI B-545 and are uncorrected.

Compound preparation and characterization:
Synthesis of 3 methanaminium chloride (3) To a suspension of lithium aluminium hydride (117 mg, 3.07 mmol) in anhydrous diethyl ether (6 mL), under nitrogen, was added aluminium chloride (408 mg, 3.07 mmol). The reaction mixture was cooled to 0 °C and stirred for 10 min. The reaction was allowed to warm to rt and the 3-chloro-4- Tetrakis(triphenylphosphine)-palladium (1.27 g, 1.10 mmol) was added and the mixture was heated to reflux for 3.5 h. The mixture was allowed to cool to rt and poured into water, then extracted with ethyl acetate (3x). The combined organic extracts were dried (MgSO 4 ) and the solvent evaporated under reduced pressure. The crude residue was purified by column chromatography eluting with a 98:2 to 90:10 n-hexane/ethyl acetate gradient to yield the product as a white solid (3.69 g, 77%).