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

蛋白激酶是细胞中最重要的控制酶之一;它们利用ATP将磷酸基团转移到其底物蛋白上，从而调节其活性。突变或失调的蛋白激酶积极参与肿瘤的发展，因此是新的癌症治疗的最重要的分子靶标之一。蛋白激酶CK 2是用于开发针对许多肿瘤类型的新药的经验证的靶蛋白。一种结合并阻断ATP结合口袋的抑制剂（CX-4945）目前正在临床试验中，并首次显示出抗肿瘤作用。然而，它对CK 2没有特异性，但在相同程度上抑制其他三种蛋白激酶，这可能导致剂量限制性副作用。缺乏选择性是由于与ATP口袋的结合，这在所有500多种人类蛋白激酶中非常相似。相反，靶向替代结合位点的变构抑制剂通常更具选择性。我们的小组最近发现了新的蛋白激酶CK 2的变构抑制剂，其结合到称为“P环”口袋的替代结合位点。这些已被证明对CK 2更具选择性，并且在抑制细胞中的CK 2方面非常有效。不同的变构抑制机制也导致化合物以不同的强度抑制不同的底物，因为变构机制也可以被反调节。在与CX-4945的直接比较中，我们的抑制剂在肿瘤细胞中比在非肿瘤细胞中更有选择性地触发程序性细胞死亡。在未来的治疗应用中，这可能意味着更高的肿瘤选择性，相应地具有更低的副作用，然而，这需要在使用动物模型的先前研究中进行测试。因此，我们研究项目的主要目标是在效力、选择性和代谢稳定性方面改善现有的铅结构，使其适合在动物模型中进行首次测试（计划作为后续研究的一部分）。分子部分将被单独优化，获得的最佳抑制剂将与CK 2蛋白共结晶，以阐明复合物的3D结构。对于CK 2的一些底物蛋白，已知它们与激酶形成复合物，这使得特别有效的磷酸化成为可能。蛋白质之间的接触位点与我们的变构抑制剂的结合位点重叠;因此，作为一种额外的效应，我们的抑制剂的结合也可能从与CK 2的复合物中置换底物蛋白，并可持续地防止再磷酸化。这种有利的额外效果将在新的衍生产品中得到特别的增强。将开发新的基于荧光的细胞测定法，以进一步分析和量化这种影响。

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.