A fragment-based approach leading to the discovery of inhibitors of CK2α with a novel mechanism of action

CK2 is a ubiquitous protein kinase with an anti-apoptotic role and is found to be overexpressed in multiple cancer types. To this end, the inhibition of CK2 is of great interest with regard to the development of novel anti-cancer therapeutics. ATP-site inhibition of CK2 is possible; however, this typically results in poor selectivity due to the highly conserved nature of the catalytic site amongst kinases. An alternative methodology for the modulation of CK2 activity is through allosteric inhibition. The recently identified αD site represents a promising binding site for allosteric inhibition of CK2α. The work presented herein describes the development of a series of CK2α allosteric inhibitors through iterative cycles of X-ray crystallography and enzymatic assays, in addition to both fragment growing and fragment merging design strategies. The lead fragment developed, fragment 8, exhibits a high ligand efficiency, displays no drop off in activity between enzymatic and cellular assays, and successfully engages CK2α in cells. Furthermore, X-ray crystallographic analysis provided indications towards a novel mechanism of allosteric inhibition through αD site binding. Fragments described in this paper therefore represent promising starting points for the development of highly selective allosteric CK2 inhibitors.

efficiencies. % inhibitions are given as the mean ± SEM from 3 repeats; IC 50 's are given as the mean ± SEM from 3 repeats; assays were run with CX4945 as a positive control and DMSO as a negative control; ND = not determined; *under deposition currently.

Chemistry experimental
All experiments were carried out in oven-dried glassware under an atmosphere of N 2 using distilled solvents unless otherwise stated.
Reagents: Chemicals were purchased from commercial sources and used without further purification.
Yield: refers to chromatographically and spectroscopically pure compounds unless otherwise stated and are reported as follows: mass, moles, percentage.
Temperature: Reaction temperatures of 0 °C were maintained using an ice-water bath; room temperature (rt) refers to 20-25 °C.

Protein expression and purification
Expression and purification of CK2α (wild-type, KKK/AAA and SF) was performed as previously described. 5

Isothermal titration calorimetry
All ITC experiments were performed at 25 °C using a MicroCal iTC200 instrument (GE Healthcare). CK2αWT

X-ray crystallography
Crystallization, soaking of ligands, and structure determination were done as described before. X-ray diffraction data were collected at the Diamond Light Source and data from automated data processing with autoProc were used for the structure determination. 6 All coordinates have been deposited to the Protein Data Bank under the accession numbers in the table below.

Cell viability assay
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 flatbottomed 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  Membranes were then incubated with either anti-AKT1 (phosphoS129) (Abcam, ab133458) or anti-Cdc37 (phospho S13) (Abcam, ab108360) antibodies diluted in 5% BSATBST 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 reprobing, membranes were immersed in Restore Western Blot stripping buffer (Thermo Scientific) for 15 minutes at room temperature.

Modelling
Modelling was performed using Glide. The modelling was based upon PDB: 7ZY2, the binding site was defined as a 10 Angstrom sphere around compound 3 bound in the αD site. Ligand structures were generated using Knime and results were visualised in Schrodinger.