PowerPoint Presentation

Specific inhibition of CK2 from outside the active site:A specific CK2a inhibitor utilising a cryptic D pocket

13/05/2016

Paul Brear

CK2- A promising target for cancer therapy CK2 is a highly conserved, constitutively active Ser/Thr kinase with over 300 target.

CK2 is often overexpressed many in cancer, promoting their proliferation by multiple mechanisms.

CK2 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)

EnzymeIC50ReferenceCK214.7 nMPLOS ONE (2014) 9:4CLK182 nMPLOS ONE (2014) 9:4CLK23.8 nMPLOS ONE (2014) 9:4CLK390 nMPLOS ONE (2014) 9:4CK21 nMJ.Med. Chem. (2011) 54:2:635DAPK317 nMJ.Med. Chem. (2011) 54:2:635TBK135 nMJ.Med. Chem. (2011) 54:2:635FLT355 nMJ.Med. Chem. (2011) 54:2:635PIM146 nMJ.Med. Chem. (2011) 54:2:635PIM148 nMbiorg. Med. Chem. Lett. (2011) 21;22;6687CLK341 nMJ.Med. Chem. (2011) 54:2:635CDK156 nMJ.Med. Chem. (2011) 54:2:635HIPK345 nMJ.Med. Chem. (2011) 54:2:635PIM2186 nMbiorg. Med. Chem. Lett. (2011) 21;22;6687CDK21800 nMEur. J. Med. Chem. (2014) 78:217-224

More selective inhibitors of CK2 clearly have great potential as therapeutic agents and chemical tools.

The most validated CK2 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 CK2 inhibitors

Aims AimDevelop selective inhibitors of CK2 that bind outside of the conserved ATP site.

7) Validation ofnovel inhibitorAffinity InhibitionSelectivityGI50 against cancer cell lines Target engagement in cells

Fragments were screened against CK2 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 CK2

NMR154L

The D loop in CK2 has very low conservation across kinases therefore targeting this site is likely to lead to high selectivity for CK2

The D site

Aims AimDevelop selective inhibitors of CK2 that bind outside of the conserved ATP site.

7) Validation ofnovel inhibitorAffinity InhibitionSelectivityGI50 against cancer cell lines Target engagement in cells

Aims AimDevelop selective inhibitors of CK2 that bind outside of the conserved ATP site.

7) Validation ofnovel inhibitorAffinity InhibitionSelectivityGI50 against cancer cell lines Target engagement in cells

Elaboration of fragment core Aims 1) Increase the selectivity for the D site over the ATP site. 2) Increase affinity for the D site.

Kd = 280 M

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 siteD site

Aims AimDevelop selective inhibitors of CK2 that bind outside of the conserved ATP site.

7) Validation ofnovel inhibitorAffinity InhibitionSelectivityGI50 against cancer cell lines Target engagement in cells

Identification of ATP site warhead

Highest affinity fragmentSelected fragment

ATP siteD site

Aims AimDevelop selective inhibitors of CK2 that bind outside of the conserved ATP site.

7) Validation ofnovel inhibitorAffinity InhibitionSelectivityGI50 against cancer cell lines Target engagement in cells

Optimisation of the linker

ATP siteD site

Position of surface of channel in apo structure

Aims AimDevelop selective inhibitors of CK2 that bind outside of the conserved ATP site.

7) Validation ofnovel inhibitorAffinity InhibitionSelectivityGI50 against cancer cell lines Target engagement in cells

Linked compound (CAM4066)

The optimised fragment was linked to the selected ATP site fragment using the information from the linker optimisation. CAM4066

Aims AimDevelop selective inhibitors of CK2 that bind outside of the conserved ATP site.

7) Validation ofnovel inhibitorAffinity InhibitionSelectivityGI50 against cancer cell lines Target engagement in cells

IC50 = 366 nM Validation of CAM4066

Luciferase based kinase assay

CAM406646

Selectivity of CAM4066

This work was performed by Dr Nicola-Jane Francis in Ashok Venkitaramans lab at the MRC research centre

GI50 (M)CompoundA549HCT116JurkatCX49451544proCAM40662075

Validation of pro-CAM4066

pro-CAM4066

pro-CAM4066Validation of pro-CAM4066

Aims AimDevelop selective inhibitors of CK2 that bind outside of the conserved ATP site.

ConclusionA novel binding site close to the ATP site has been identified. CAM4066 a High affinity inhibitor that links the D site with the ATP site has been developed by linking fragments in the ATP and D site. Good Selectivity by CAM4066 has been demonstrated for CK2 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.

Acknowledgements Biochemistry Marko Hyvonen

Hyvonen group

Dimi Chiragadze Katherine Stott

Chemistry David Spring Claudia De-Fusco Kathy Hadje-GeorgiouHannah F. Sore

Chris Abell

Cell BiologyNicola-Jane FrancisAshok Venkitaraman

Loop movement

Selectivity of CAM4066 0.5 M0.5 M2 MGini Coefficient = 0.615Gini Coefficient = 0.755Gini Coefficient = 0.82

EnzymeIC50ReferenceCK214.7 nMPLOS ONE (2014) 9:4CLK182 nMPLOS ONE (2014) 9:4CLK23.8 nMPLOS ONE (2014) 9:4CLK390 nMPLOS ONE (2014) 9:4CK21 nMJ.Med. Chem. (2011) 54:2:635DAPK317 nMJ.Med. Chem. (2011) 54:2:635TBK135 nMJ.Med. Chem. (2011) 54:2:635FLT355 nMJ.Med. Chem. (2011) 54:2:635PIM146 nMJ.Med. Chem. (2011) 54:2:635PIM148 nMbiorg. Med. Chem. Lett. (2011) 21;22;6687CLK341 nMJ.Med. Chem. (2011) 54:2:635CDK156 nMJ.Med. Chem. (2011) 54:2:635HIPK345 nMJ.Med. Chem. (2011) 54:2:635PIM2186 nMbiorg. Med. Chem. Lett. (2011) 21;22;6687CDK21800 nMEur. J. Med. Chem. (2014) 78:217-224

Selectivity of CAM4066