A Biochemical Approach To Discriminate Between Atp-Competitive And Non-Atp Competitive Protein Kinase Inhibitors

Protein kinases are central to cellular signal transduction and regulation of cellular processes, and are one of the most attractive target classes in modern drug discovery. Multiple kinase inhibitors have already been approved for treatment of various diseases including such severe conditions as cancer. The first and up to today largest group of drugs that effectively inhibits their respective kinase targets belong to the class of ATP competitive compounds. They bind into or near the ATP binding site of the enzymes and inhibit kinase activity by blocking access of ATP to the active site. Although there are numerous examples of highly specific ATP competitive compounds this mode of action is limited by several factors: The ATP binding pocket structures of kinases show a high degree of similarity, which makes finding highly selective compounds challenging. Furthermore, competing with ATP for binding to the same target site, compounds have to be of very high affinity due to the high intracellular ATP concentrations. Therefore, the interest to develop non-ATP competitive inhibitors has risen considerably over the last years. Such inhibitors bind to kinases at sites apart from the ATP binding site, inhibiting their activity e.g. by stabilizing an inactive conformation (like DFG-out state binders), displacing essential cofactors (like cyclins for CDKs) or by blocking activating modifications (like phosphorylation by upstream kinases). We present data of an in-vitro biochemical kinase activity assay setup which is suited to discriminate between ATP-competitive and non-ATP competitive inhibitors. The IC 50 of an ATP-competitive inhibitor will increase with increasing ATP concentrations and the IC 50 value at a given ATP concentration may be calculated using the equation of Cheng and Prussof: IC 50 =Ki+(Ki*[ATP]/K M [ATP] (Cheng Y., Prusoff W. H. (1973) Biochem. Pharmacol. 22: 3099-3108). By determining IC 50 values for an inhibitor of a specific kinase at different ATP concentrations we examined whether the IC 50 value changed according to the Cheng-Prusoff equation, indicating an ATP-competitive mode of action, or if the IC 50 values remained unchanged in presence of elevated ATP indicating a non-ATP competitive or mixed type mode of action. In our assay setup we determined the IC 50 values at ATP concentrations in a range of 0.1 to 10 fold the ATP K M of the kinase of interest. By comparing the results obtained for the non-ATP competitive MEK1 inhibitor selumetinib and the ATP competitive inhibitor staurosporine we could verify that our assay setup is well suited to discriminate between these different types of kinase inhibitors. Citation Format: Daniel Mueller, Frank Totzke, Thomas Weber, Carolin Heidemann-Dinger, Constance Ketterer, Diane Kramer, Marcel Pathe, Michael H. Kubbutat. A biochemical approach to discriminate between ATP-competitive and non-ATP competitive protein kinase inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4186. doi:10.1158/1538-7445.AM2017-4186