Development of allosteric inhibitors of protein kinase CK2 with additional inhibitory effect on protein-protein interactions

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Protein kinases are among the most important control enzymes in the cell; they use ATP to transfer a phosphate group to their substrate proteins, thereby regulating their activity. Mutated or dysregulated protein kinases are actively involved in tumor development and are therefore among the most important molecular targets for new cancer therapeutics. The protein kinase CK2 is a validated target protein for the development of new drugs against many tumor types. An inhibitor (CX-4945) that binds to and blocks the ATP binding pocket is currently in clinical trials and has shown first antitumour effects. However, it is not specific for CK2, but inhibits three other protein kinases to the same extent, which could lead to dose-limiting side effects. The lack of selectivity is due to the binding to the ATP pocket, which is very similar in all of the more than 500 human protein kinases. In contrast, allosteric inhibitors that target alternative binding sites are usually more selective. Our group has recently discovered new allosteric inhibitors of protein kinase CK2 that bind to an alternative binding site called "P-loop" pocket. These have been shown to be more selective for CK2 and very effective in inhibiting CK2 in cells. The different allosteric inhibition mechanism also causes the compounds to inhibit different substrates with varying strength, as allosteric mechanisms can also be counterregulated. In a direct comparison with CX-4945, our inhibitors triggered programmed cell death more selectively in tumor cells than in non-tumor cells. In future therapeutic applications, this could mean higher tumor selectivity with correspondingly lower side effects, which, however, needs to be tested in prior studies using animal models. The main goal of our research project is therefore to improve the existing lead structures in terms of potency, selectivity and metabolic stability so that they are suitable for a first test in animal models (planned as part of a follow-up study). The molecule parts will be individually optimized, and the best inhibitors obtained will be co-crystallized with CK2 protein to elucidate the 3D structure of the complex. For some substrate proteins of CK2 it is known that they are found in complexes with the kinase, which enables particularly efficient phosphorylation. The contact site between the proteins overlaps with the binding site of our allosteric inhibitors; hence, as an additional effect, binding of our inhibitors might also displace the substrate proteins from the complex with the CK2 and sustainably prevent rephosphorylation. This favorable additional effect will be specifically enhanced in the new derivatives. New fluorescence-based cell assays will be developed to further analyze and quantify this effect.