Second-generation CK2α inhibitors targeting the αD pocket

We describe the development of a CAM4712, a novel CK2α inhibitor which does not interact with the ATP binding site and shows improved properties over the first-generation inhibitor CAM4066.


Table of contents
Protein expression and purification 11 X-ray crystallography 11 Growth inhibition assay 12 Western blotting 13 General synthetic experimental details 14 General synthetic methods 16 Compound overview scheme 19 Compounds and characterisation 21 Compound spectra 57 Table S1: Enthalpic and entropic contribution to Kd 122 Table S2: Crystallisation and soaking conditions 124 Table S3  125 3 FIGURE S1 Figure S1a The crystal structure showing the two binding modes of 7 in the αD site of CK2α. The hydrogen bonding between the OH group and a conserved water in the Tyr125 binding pocket of CK2α are highlighted.

FIGURE S4
Dose-response curves of compounds 22, 23, 26, 29 and CAM4712 for CK2 inhibition. It should be noted that we the exception of compound 22, the compounds were not soluble under the assay conditions, at concentration greater than 50 µM and therefore 100% inhibition was not achieved. All graphs show the mean ± SEM of not less than three independent experiments with each in triplicate.

FIGURE S5
Dose-response curves of compounds 23, 26, 29 for anti-proliferative inhibition of HCT116 cells. All graphs show the mean ± SEM of not less than three independent experiments with each in triplicate.

FIGURE S6
a) 37 into CK2α. i) The crystal structure of 37 bound in the αD pocket of CK2α. ii) The binding isotherm of 37 titrated into CK2α. b) 37 into CK2α and 20 µM CAM4712. i) The crystal structure of CAM4712 showing how it binds to CK2α. ii) The binding isotherm of 37 titrated into CK2α and 20 µM CAM4712 binding in the αD site. The presence of CAM4712 does not significantly change the affinity of 37 for the αD site. c) The structure of CAM4712 (light blue) bound in the αD site with the structure of 37 (purple) bound in the ATP site superimposed. d) The cocrystal structure of CAM4712 and 37 binding simultaneously to CK2α. The Fo-Fc map is shown contoured at 1.6σ.
ii) The binding isotherm of CX4945 titrated into CK2α binding in the ATP site. b) CX4945 into CK2α and 20 µM CAM4712. i) The crystal structure of CAM4712 showing how it binds to CK2α. ii) The binding isotherm of CX4945 titrated into CK2α and 20 µM CAM4712. The presence of CAM4712 reduces the apparent affinity of CX4945 for the αD site. c) The structure of CAM4712 (light blue) bound in the αD site with the structure of CX4945 (green) bound in the ATP site superimposed. d) The structure of CAM4712 (light blue) bound in the αD site with the structure of CX4945 (green) and 37 bound in the ATP site superimposed.

FIGURE S8
A) Selectivity profile of CAM4712 at 30 µM concentration against closely related CMGC family members; B) Selectivity profile of CAM4712 at 30 µM against a panel of 140 kinases. Kinases inhibited by CAM4712 for more than 50% are highlighted in red. Kinases inhibited by other CK2 kinase inhibitors (CX4945, D11, TF, CX5279, 7h) are shown as coloured dots.

Protein expression and purification
Three constructs of CK2α were used in this study. For ITC and kinase activity assays CK2α_WT was used (residues 2-329). For crystallization purposes two different constructs were used: CK2α_KA and CK2α_FP10. 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_FP 10 contained one mutation (R21S) and an N-terminal extension GSMDIEFDDDADDDGSGSGSGSGS aimed at mimicking a substrate peptide for CK2α. CK2α_FP10 was cloned into pHAT4 vector and CK2α_KA was cloned into pHAT2 vector to give constructs with cleavable His6-tags. Recombinant plasmids containing one of the three constructs (CK2α_WT/ CK2α_KA/ CK2α_FP10) were introduced into Escherichia coli BL21(DE3) for protein production. Single colonies of the cells were grown in 6x1L of 2xTY with 100 μg/mL ampicillin at 37°C. Isopropylthio-β-D-galactopyranoside (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 required 500 mM NaCl. The cell pellets were suspended in 20 mM Tris, 350/50 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 N-terminal His6-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 cryocooled in liquid nitrogen in the same solution for data collection. The crystals were cryo-cooled in liquid nitrogen in the same solution for data collection. X-ray diffraction data was collected at the Diamond synchrotron radiation source, then processed using the pipedream package by Global Phasing Ltd; structures were solved by using programs from the CCP4 package. Models were iteratively refined and rebuilt by using AutoBuster and Coot programs. Ligand coordinates and restraints were generated from their SMILES strings using the Grade software package. All coordinates have been deposited to Protein Data Bank and accession numbers, data collection and refinement statistics are shown in Table S3, with crystallisation and soaking conditions being listed in Table S2.

ITC
All ITC experiments were performed at 25 °C using a MicroCal itc200 instrument (GE Healthcare). CK2α_WT (20 mg/mL, 20 mM 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 MgCl2, 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 IC50 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%. All cells were grown at 37°C / 5% CO2 in a humidified environment and all the assays were performed using these culturing conditions. Growth Inhibition assays Adherent cell lines (HCT116) 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. 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. Without removing supernatant 100 μL of cold 10% (v/v) trichloroacetic acid was added to each well and the plates were incubated for 30 minutes at 4 °C. After that 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 200 μL 10 mM Tris solution (pH 10.5) and incubated for 30 minutes under agitation. The 510 nm absorbance was then measured using a PHERAstar plus plate reader (BMG Labtech). Percentage of growth inhibition was calculated relative to DMSO controls and GI50 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, 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 NC membrane at 4°C overnight at a constant 12 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% Tween20) for 1 hour. Membranes were then incubated with anti-AKT1 (phosphoS129) (Abcam, ab133458) antibodies diluted 1:3.000 in 5% BSA-TBST 0.1% Tween20 for 24 hours at 4 °C. Anti-GAPDH antibody was used as a loading control (Sigma-Aldrich, G8795) diluted in Milk-TBS-0.1% Tween20. After washing, membranes were then incubated with HRP-labelled anti-rabbit or anti-mouse antibody for 1 hour at room temperature, washed, and then visualised using ECL Clarity TM (Bio-Rad).

GENERAL SYNTHETIC 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 CaH2 and LiAlH4 in the presence of triphenylmethane. Diethyl ether was distilled from CaH2 and LiAlH4. CH2Cl2 and MeOH were distilled from CaH2. All other solvents were used as obtained from commercial sources.
Materials: All reagents were used as obtained from commercial sources.
TLC: All reactions were monitored by thin layer chromatography (TLC) using glass plates precoated with Merck silica gel 60 F254. Visualization was by the quenching of UV fluorescence (λmax = 254 nm) or by staining with ninhydrin. Retention factors (Rf) are quoted to 0.01.
Chromatography: Flash column chromatography was carried out using slurry-packed Merck 9385 Kieselgel 60 silica gel under a positive pressure of nitrogen.

NMR:
Magnetic resonance spectra were processed using iNMR v. 5.5.7 (Mestrelab Research) or TopSpin v. 3.5 (Bruker). An aryl, quaternary, or two or more possible assignments were given when signals could not be distinguished by any means. Measured coupling constants are reported for mutually coupled signals; coupling constants are labelled apparent in the absence of an observed mutual coupling, or multiplet when none can be determined.
Proton magnetic resonance spectra were recorded using an internal deuterium lock (at 298 K unless stated otherwise) on Bruker DPX (400 MHz; 1H-13C DUL probe), Bruker Avance III HD (400 MHz; Smart probe), Bruker Avance III HD (500 MHz; Smart probe) and Bruker Avance III HD (500 MHz; DCH Cryoprobe) spectrometers. Proton assignments are supported by 1 H-1 H COSY, 1 H-13 C HSQC or 1 H-13 C HMBC spectra, or by analogy. Chemical shifts (δH) are quoted in ppm to the nearest 0.01 ppm and are referenced to the residual non-deuterated solvent peak. Discernible coupling constants for mutually coupled protons are reported as measured values in Hertz, rounded to the nearest 0.1 Hz. Data are reported as: chemical shift, multiplicity (br, broad; s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; or a combination thereof), coupling constants and number of nuclei. Diastereotopic protons are assigned as X and X', where X' designates the lower-field proton.
Carbon magnetic resonance spectra were recorded using an internal deuterium lock (at 298 K unless stated otherwise) on Bruker DPX (101 MHz), Bruker Avance III HD (101 MHz) and Bruker Avance III HD (126 MHz) spectrometers with broadband proton decoupling. Carbon spectra assignments are supported by DEPT editing, 1 H-13 C HSQC or 1 H-13 C HMBC spectra, or by analogy. Chemical shifts (δC) are quoted in ppm to the nearest 0.1 ppm and are referenced to the deuterated solvent peak. Data are reported as: chemical shift, multiplicity (if not a singlet), coupling constants and number of nuclei (if not one).
Fluorine magnetic resonance spectra were recorded on Bruker Avance III (376 MHz; QNP Cryoprobe) or Bruker Avance III HD (376 MHz; Smart probe) spectrometers. Chemical shifts (δF) are quoted in ppm to the nearest 0.1 ppm. Data are reported as: chemical shift, number of nuclei (if not one), multiplicity (if not a singlet), coupling constants and assignment.

HRMS:
High resolution mass spectrometry was carried out with a Micromass Q-TOF or a Waters LCT Premier Mass Spectrometer using electrospray ionisation [ESI].
Melting points: These data were collected on a BÜCHI B-545 and are uncorrected.

General method A: Phenol triflation
To a solution of phenol (1.0 equiv) in anhydrous CH2Cl2 (~0.3 M) was added pyridine (1.6 equiv). The solution was cooled to 0 °C and trifluoromethanesulfonic anhydride (1.4 equiv) was added dropwise over 30 minutes. The reaction was allowed to warm to room temperature and stirred overnight. The volatiles were removed under reduced pressure and the residue was diluted with H2O and extracted with EtOAc. The organic layer was washed with 10% aqueous HCl, 5% aqueous Na2CO3, a saturated aqueous solution of NaCl and H2O, then dried over MgSO4, filtered and concentrated in vacuo. The crude product was then purified by flash column chromatography to yield the desired product.
General method B: Suzuki-Miyaura coupling 1 A mixture of the aryl bromide (1.0 equiv.) or aryl triflate (1.0 -1.6 equiv), appropriate boronic acid (1.0 -1.3 equiv), Pd(PPh3)4 (0.16 -0.05 equiv) and 2M aqueous Na2CO3 (1.6 -3.0 equiv) or K2CO3 (2.0 equiv) were solvated with DME (0.16 -0.7 M) and H2O (1.4 -2.3 M). The reaction was degassed by bubbling nitrogen through the solution for 15 minutes and then heated to reflux or heated to 100 o C under microwave irradiation until consumption of the starting material (1-7 hours) by TLC monitoring. The reaction was allowed to cool to room temperature, filtered through celite washing with Et2O and the solvent removed under reduced pressure. The residue was dissolved in CH2Cl2/H2O and extracted three times with CH2Cl2. The combined organic extracts were washed with a saturated aqueous solution of NaCl, dried over MgSO4, filtered and concentrated in vacuo. The crude product was then purified by flash column chromatography to yield the desired product.
General method C: Suzuki-Miyaura coupling 2 A mixture of the aryl bromide (1.0 -1.2 equiv) or aryl triflate (1.0 equiv.), appropriate boronic acid (1.0 -1.2 equiv), PdCl2 (dppf)·CH2Cl2 (0.05 equiv) and K3PO4 (1.20 -2.0 equiv) were solvated with DME (0.2 -0.3 M), EtOH (1.3 -1.7 M) and H2O (2.0 -2.5 M). The reaction mixture was degassed by bubbling nitrogen through the solution for 15 minutes and then heated to reflux or heated to 110 o C under microwave irradiation until consumption of the starting material (1-6 hours) by TLC monitoring. The reaction was allowed to cool to room temperature, filtered through celite washing with Et2O and the solvent removed under reduced pressure. The residue was dissolved in Et2O/H2O and extracted three times with Et2O. The combined organic extracts were washed with a saturated aqueous solution of NaCl, dried over MgSO4, filtered and concentrated in vacuo. The crude product was then purified by flash column chromatography to yield the desired product.
General method D: Suzuki-Miyaura coupling 3 A mixture of the benzaldehyde (1.0 equiv), appropriate boronic acid (2.0 equiv) and K3PO4 (4.0 equiv) was dissolved in 1,4-dioxane (0.25 M). The reaction mixture was degassed by bubbling nitrogen through the solution for 15 minutes before the addition of PdCl2(dppf) (0.10 equiv). The reaction mixture was then heated to 90 o C until consumption of the starting material (2.5-6 hours) by TLC monitoring. The mixture was then diluted with EtOAc and washed twice with H2O. The organic layer was washed with a saturated aqueous solution of NaCl, dried over MgSO4, filtered and concentrated in vacuo. The crude product was then purified by flash column chromatography to yield the desired product.
General method E: Benzonitrile reduction To a stirred suspension of LiAlH4 (2.0 -4.0 equiv) in Et2O (~0.25 M) was added AlCl3 (0.0 -4.0 equiv) and the reaction mixture cooled to 0 °C for 10 minutes. The reaction was allowed to warm to room temperature and the nitrile (1.0 equiv) was added portionwise. The reaction was stirred at room temperature for 30 minutes and then heated at 50 °C overnight. After cooling to room temperature, a saturated aqueous solution of potassium sodium tartrate tetrahydrate and Et2O were added and the mixture stirred for 1 hour. The reaction mixture was diluted with 2M aqueous NaCO3 and extracted three times with Et2O. The combined organic extracts were washed with a saturated aqueous solution of NaCl, dried over MgSO4, filtered and concentrated in vacuo. The crude product was then purified by flash column chromatography or semi-preparative HPLC to yield the desired product.
General method F: Reductive amination of free amines The benzaldehyde (1.0 equiv) and the amine (1.3 -1.5 equiv) were combined in anhydrous 1,2dichloroethane (0.28 M) under an atmosphere of nitrogen and stirred for 2 hours. Sodium triacetoxyborohydride (1.4 equiv) was added in two portions with a 30 minute interval and the reaction was stirred at room temperature for 18 hours. The reaction mixture was poured into 2M aqueous Na2CO3 and extracted three times with CH2Cl2. The combined organic extracts were washed with a saturated aqueous solution of NaCl, dried over MgSO4, filtered and concentrated in vacuo. The crude product was then purified by flash column chromatography to yield the desired product.
General method G: Reductive amination of ammonium salts A solution of the ammonium salt (1.5 equiv) in MeOH (0.28 M) was treated with NEt3 (2.0 equiv) and aldehyde (1.0 equiv) and the mixture stirred at room temperature for 2 hours. Sodium triacetoxyborohydride (1.4 equiv) was added in two portions with a 30 minute interval and the reaction was stirred at room temperature for 18 hours. The reaction mixture was poured into 2 M aqueous Na2CO3 and extracted three times with CH2Cl2. The combined organic extracts were washed with a saturated aqueous solution of NaCl, dried over MgSO4, filtered and concentrated in vacuo. The crude product was then purified by flash column chromatography to yield the desired product.

General method H: Benzimidazole condensation
The appropriate carboxylic acid (1.0 equiv), diamine (2.0 equiv), HBTU (2.0 equiv) and NEt3 (3.0 equiv) were combined in anhydrous DMF (0.09 M) under at atmosphere of nitrogen and stirred for 2.5 hours. The reaction mixture was diluted with EtOAc and washed with H2O, a saturated aqueous solution of NaHCO3 and a saturated aqueous solution of NaCl before drying over Na2SO4, filtration and concentrated in vacuo. The crude product was purified by flash column chromatography and the resulting residue dissolved in acetic acid (0.09 M) and heated to 70 °C overnight. Following cooling to 0 °C, the reaction mixture was poured into a saturated aqueous solution of NaHCO3 and extracted three times with EtOAc. The combined organic extracts were washed with a saturated aqueous solution of NaCl, dried over Na2SO4, filtered and concentrated in vacuo. The crude product was then purified by flash column chromatography to yield the N-boc-protected product.

General method J: Boc-deprotection and salt formation
A solution of protected amine (1.0 equiv) in HCl (4M in 1,4-dioxane) (~100 equiv) was stirred at room temperature for 3 hours. The volatiles were removed in vacuo and the residue triturated with Et2O to yield the desired product.