Abstract 5168: Effects of point mutations, recombinant tags, activation status, and identity of target kinases on the mode of action of approved kinase inhibitors.
Authors: Müller Daniel , Beisenherz-Huss Christian , Totzke Frank , Heidemann-Dinger Carolin , Ketterer Constance , Weber Thomas , Kubbutat Michael H.G. 
Published: August 2014

Abstract
Protein kinases regulate a variety of biological signaling pathways affecting cell proliferation, differentiation, migration and apoptosis. In tumor cells the activity of many protein kinases are frequently found to be upregulated linking deregulation of protein kinases causally to the development and progression of many human cancers and other diseases.
Therefore, protein kinases have become a prime molecular target for therapeutic intervention, and up to now fourteen small molecule inhibitors have been approved for treatment of various types of cancer. These inhibitors block the activity of the target protein kinases by either blocking the ATP binding site in a direct competitive manner (type1-inhibitors) or by indirectly interfering with ATP/kinase interaction by binding to an inactive state, referred to as DFG-out (type2-inhibitors). Due to the fact that these inhibitor types bind to a region which is highly conserved among the protein kinase superfamily, achieving target selectivity represent a major challenge in the development of protein kinase inhibitors. Allosteric inhibitors, also sometimes referred to as type3-inhibitors, bind to structural sites different to the ATP-binding pocket region, and have, therefore, a significant higher potential to inhibit kinases much more selective than ATP-competitive compounds.
The majority of the currently clinically approved small molecule protein kinase inhibitors are commonly classified as ATP-competitive inhibitors, and are able to inhibit different protein kinases with high potency. Interestingly, analysing the effect of the ATP-concentration on the IC50 of Sorafinib against different target kinases, we observed that the inhibitory potency against various target kinases is differently affected by the ATP concentration indicating differences in the exact mode of action of the inhibitor. To answer the question whether this is a specific property of Sorafenib, or also relevant for other inhibitors, we perfomed similar studies with seven additional clinically approved kinase inhibitors (Axitinib, Crizotinib, Erlotinib, Gefitinib, Lapatinib, Pazopanib and Sunitinib) using a panel of up to 16 different target protein kinases. We extended these studies and will in addition also present data investigating the influences of (a) the tags to which the respective recombinant kinase was fused, (b) the activation status of the kinase, and (c) of activating point mutations on the mode of action of the different inhibitors.
Citation Format: Daniel Müller, Christian Beisenherz-Huss, Frank Totzke, Carolin Heidemann-Dinger, Constance Ketterer, Thomas Weber, Michael H.G. Kubbutat. Effects of point mutations, recombinant tags, activation status, and identity of target kinases on the mode of action of approved kinase inhibitors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5168. doi:10.1158/1538-7445.AM2013-5168