18 September 2017

Fragment linking to a selective CK2 inhibitor

The kinase CK2 is an intriguing anti-cancer target, but most of the reported inhibitors bind in the conserved hinge region and so also hit other kinases, complicating interpretation of the biology. A team based at the University of Cambridge has taken a fragment-linking approach to discover more selective inhibitors. The first report was published last year by Marko Hyvönen, David Spring, and colleagues in Chem. Sci., and they have now published a more complete account in Bioorg. Med. Chem.

A crystallographic screen identified compound 1, which bound to six different sites! One of these sites was particularly interesting as it appeared to be a previously undiscovered “αD” pocket near the ATP-binding site. A couple cycles of SAR by catalog, informed by computational screening, led to compound 7, which binds in the desired pocket but not at other sites.

Although compound 7 has measureable affinity for CK2α as judged by ITC, it does not inhibit the enzyme, which is not surprising because it does not bind in the ATP-binding site. Thus, the researchers screened 352 fragments from Zenobia in cocktails of 4, each at 5 mM, and found 23 that bound in the ATP site. Reasoning that the hinge region is the most conserved portion of the ATP-binding site, the researchers avoided fragments that bound there. This led them to focus on compound 8, which has a synthetic handle pointing towards the αD pocket.

Next, modeling was used to generate a series of appendages from compound 7 to try to reach compound 8. Compound 19 looked like it could bridge the gap, a hypothesis which was confirmed when linking led to a low micromolar binder. Tweaking the linker led to CAM4066, which showed nanomolar binding as well as inhibition of CK2. Crystallography revealed that the linked molecule bound as expected.

CAM4066 was tested against 52 other kinases at 2 µM and showed at most only 20% inhibition, suggesting that it is indeed quite selective for CK2. Unfortunately, perhaps because of its carboxylic acid, it did not show any cell activity. This was addressed by making a methyl ester prodrug – a strategy that was also taken for another fragment linking campaign on a very different target.

As the researchers point out, CAM4066 follows the Evotec model of a largely lipophilic fragment linked to a more polar fragment. There is still much more to do: no pharmacokinetic data are provided, and the potency still falls short of what is needed for a chemical probe. Still, this is a nice illustration of the power of fragment linking, guided by both modeling and crystallography, to generate molecules with interesting properties.

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