The development of selective inhibitors of protein kinases is challenging because of the significant conservation of the ATP binding site. Here, we describe a new mechanism by which the protein kinase CK2α can be selectively inhibited using features outside the ATP site. We have identified a new binding site for small molecules on CK2α adjacent to the ATP site and behind the αD loop, termed the αD pocket. An elaborated fragment anchored in this site has been linked with a low affinity fragment binding in the ATP site, creating a novel and selective inhibitor (CAM4066) that binds CK2α with a Kd of 320 nM and shows significantly improved selectivity compared to other CK2α inhibitors. CAM4066 shows target engagement in several cell lines and similar potency to clinical trial candidate CX4945. Our data demonstrate that targeting a poorly conserved, cryptic pocket allows inhibition of CK2α via a novel mechanism, enabling the development of a new generation of selective CK2α inhibitors.

1. Specific inhibition of CK2α from outside the
active site:
A specific CK2a inhibitor utilising a cryptic αD pocket
13/05/2016
Paul Brear

2. CK2α- A promising target for cancer therapy
CK2α is a highly conserved, constitutively active Ser/Thr kinase
with over 300 target.
CK2α is often overexpressed many in cancer, promoting their
proliferation by multiple mechanisms.
CK2α inhibitors have great therapeutic potential when used
synergistically with established cancer therapies.
Interactions predicted by string
Jensen et al. Nucleic Acids Res. 2009, 37(Database
issue):D412-6
Ortega CE, Seidner Y, Dominguez I (2014) Mining CK2 in Cancer. PLoS ONE 9(12)

3. Enzyme IC50 Reference
CK2α 14.7 nM PLOS ONE (2014) 9:4
CLK1 82 nM PLOS ONE (2014) 9:4
CLK2 3.8 nM PLOS ONE (2014) 9:4
CLK3 90 nM PLOS ONE (2014) 9:4
CK2α 1 nM J.Med. Chem. (2011) 54:2:635
DAPK3 17 nM J.Med. Chem. (2011) 54:2:635
TBK1 35 nM J.Med. Chem. (2011) 54:2:635
FLT3 55 nM J.Med. Chem. (2011) 54:2:635
PIM1 46 nM J.Med. Chem. (2011) 54:2:635
PIM1 48 nM biorg. Med. Chem. Lett. (2011)
21;22;6687
CLK3 41 nM J.Med. Chem. (2011) 54:2:635
CDK1 56 nM J.Med. Chem. (2011) 54:2:635
HIPK3 45 nM J.Med. Chem. (2011) 54:2:635
PIM2 186 nM biorg. Med. Chem. Lett. (2011)
21;22;6687
CDK2 1800 nM Eur. J. Med. Chem. (2014)
78:217-224
More selective inhibitors of CK2α clearly have great potential
as therapeutic agents and chemical tools.
The most validated CK2α inhibitor
developed to date is CX-4945.
• High affinity and inhibition (IC50= 1 nM)
• Advanced to phase I/II clinical trials in
2014 against bile duct cancer.
Described as ‘highly selective’, but active
against 12 other kinases.
CX-4945
Challenges in the development of selective CK2α inhibitors

4. Aims
Aim
Develop selective
inhibitors of
CK2α that bind
outside of the
conserved ATP
site.
7) Validation of
novel inhibitor
• Affinity
• Inhibition
• Selectivity
• GI50 against cancer
cell lines
• Target engagement
in cells

5. Fragments were screened against CK2α at high
concentrations (100 mM) using X-ray
crystallography.
This lead to the discovery of a novel pocket
close to the ATP site which bound multiple
fragments.
Fragment based screen against CK2α
NMR154L

6. The αD loop in CK2α has
very low conservation
across kinases therefore
targeting this site is
likely to lead to high
selectivity for CK2α
The αD site

7. Aims
Aim
Develop selective
inhibitors of
CK2α that bind
outside of the
conserved ATP
site.
7) Validation of
novel inhibitor
• Affinity
• Inhibition
• Selectivity
• GI50 against cancer
cell lines
• Target engagement
in cells

8. Aims
Aim
Develop selective
inhibitors of
CK2α that bind
outside of the
conserved ATP
site.
7) Validation of
novel inhibitor
• Affinity
• Inhibition
• Selectivity
• GI50 against cancer
cell lines
• Target engagement
in cells

9. Elaboration of fragment core
Aims
1) Increase the selectivity
for the αD site over the
ATP site.
2) Increase affinity for the
αD site.
Kd = 280 µM
Ligand efficiency = 0.33
Selective for the Atp site.
Synthesis of analogues
All synthesis was performed by Dr
Claudia De-Fusco and Dr Kathy
Hadje-Georgiou in the group of Prof.
David Spring.
ATP site
αD site

10. Aims
Aim
Develop selective
inhibitors of
CK2α that bind
outside of the
conserved ATP
site.
7) Validation of
novel inhibitor
• Affinity
• Inhibition
• Selectivity
• GI50 against cancer
cell lines
• Target engagement
in cells

11. Identification of ATP site warhead
Highest affinity fragment
Selected fragment
ATP site
αD site

12. Aims
Aim
Develop selective
inhibitors of
CK2α that bind
outside of the
conserved ATP
site.
7) Validation of
novel inhibitor
• Affinity
• Inhibition
• Selectivity
• GI50 against cancer
cell lines
• Target engagement
in cells

13. Optimisation of the linker
ATP site
αD site
Position of surface of channel in apo structure

14. Aims
Aim
Develop selective
inhibitors of
CK2α that bind
outside of the
conserved ATP
site.
7) Validation of
novel inhibitor
• Affinity
• Inhibition
• Selectivity
• GI50 against cancer
cell lines
• Target engagement
in cells

15. Linked compound (CAM4066)
The optimised fragment was linked to the selected
ATP site fragment using the information from the
linker optimisation.
CAM4066

16. Aims
Aim
Develop selective
inhibitors of
CK2α that bind
outside of the
conserved ATP
site.
7) Validation of
novel inhibitor
• Affinity
• Inhibition
• Selectivity
• GI50 against cancer
cell lines
• Target engagement
in cells

17. IC50 = 366 nM
Validation of CAM4066
Luciferase based kinase assay
CAM4066
4 6

18. Selectivity of CAM4066

19. This work was performed by Dr Nicola-Jane Francis in
Ashok Venkitaraman’s lab at the MRC research centre
GI50 (µM)
Compound A549 HCT116 Jurkat
CX4945 15 4 4
proCAM4066 20 7 5
Validation of pro-CAM4066
pro-CAM4066

20. pro-CAM4066
Validation of pro-CAM4066

21. Aims
Aim
Develop selective
inhibitors of
CK2α that bind
outside of the
conserved ATP
site.
Conclusion
• A novel binding site close to the ATP site has
been identified.
• CAM4066 a High affinity inhibitor that links the
αD site with the ATP site has been developed
by linking fragments in the ATP and αD site.
• Good Selectivity by CAM4066 has been
demonstrated for CK2α over other kinases.
• CAM4066 has been shown to be active against
various cancer cell lines with an activity
comparable to that of the clinical candidate CX-
4945.

22. Acknowledgements
Biochemistry
Marko Hyvonen
Hyvonen group
Dimi Chiragadze
Katherine Stott
Chemistry
David Spring
Claudia De-Fusco
Kathy Hadje-Georgiou
Hannah F. Sore
Chris Abell
Cell Biology
Nicola-Jane Francis
Ashok Venkitaraman

24. Loop movement

25. Selectivity of CAM4066
-10
10
30
50
70
90
1 5 9 13 17 21 25 29 33 37 41 45 49 53 57 61 65 69 73 77 81 85 89 93 97 101
Selectivity of CX4945
-10
10
30
50
70
90
1
5
9
13
17
21
25
29
33
37
41
45
49
53
57
61
65
69
73
77
81
85
89
93
97
101
Selectivity of CX5279
0.5 µM
0.5 µM
2 µM Gini Coefficient = 0.615
Gini Coefficient = 0.755
Gini Coefficient = 0.82

26. Enzyme IC50 Reference
CK2α 14.7 nM PLOS ONE (2014) 9:4
CLK1 82 nM PLOS ONE (2014) 9:4
CLK2 3.8 nM PLOS ONE (2014) 9:4
CLK3 90 nM PLOS ONE (2014) 9:4
CK2α 1 nM J.Med. Chem. (2011) 54:2:635
DAPK3 17 nM J.Med. Chem. (2011) 54:2:635
TBK1 35 nM J.Med. Chem. (2011) 54:2:635
FLT3 55 nM J.Med. Chem. (2011) 54:2:635
PIM1 46 nM J.Med. Chem. (2011) 54:2:635
PIM1 48 nM biorg. Med. Chem. Lett. (2011)
21;22;6687
CLK3 41 nM J.Med. Chem. (2011) 54:2:635
CDK1 56 nM J.Med. Chem. (2011) 54:2:635
HIPK3 45 nM J.Med. Chem. (2011) 54:2:635
PIM2 186 nM biorg. Med. Chem. Lett. (2011)
21;22;6687
CDK2 1800 nM Eur. J. Med. Chem. (2014)
78:217-224
Selectivity of CAM4066