Protein kinases mediate many cell signaling events, and their tight control is essential for regulating vital processes ranging from cell division to energy metabolism. Thus, it is not surprising that protein kinases are directly or indirectly involved in many diseases and that kinases are key drug targets. For example, Src kinase was the first identified proto-oncogene and the formation of a de-regulated Abl fusion protein (BCRAbI) is the cause of disease in 95% of patients with chronic myeloid leukemia. X-ray crystal structures have shown that the same kinases can attain an active and various inactive conformations, implying that kinases are inherently flexible. How the active and inactive states are stabilized and how these states interconvert are key questions in understanding kinase regulation. Because X-ray crystal structures provide only static snapshots, we will use nuclear magnetic resonance (NMR) experiments and ligand binding kinetics to study the timescales and amplitudes of structural interconversions in Abl and Src kinase domains. BCR-AbI is the target of the clinically highly successful drug imatinib (Gleevec?, Novartis) in the treatment of chronic myelogenous leukemia (CML). The clinical success of imatinib is due to its excellent specificity, binding only to the inactive conformation of the kinase. Therefore drug binding is intimately related to the interconversion between active and inactive states. The goal of this study is to examine timescales and pathways ofthese interconversions between active and inactive conformations, how dynamics of structural elements relate to catalytic turnover of the kinase and how drug resistance mutations affect these dynamics. Therefore, we will compare the timescales and amplitudes of backbone motions between Src and Abl kinases in the presence of drugs by NMR experiments. Ligand binding kinetics will be used to address the role of the regulatory domains on kinase dynamics and the binding mechanisms of different classes of kinase inhibitors. The role of protein plasticity and dynamics on inhibitor promiscuity will be addressed by structural studies on kinase*inhibitor complexes, inhibitor binding kinetics and biochemical assays.
Kinase inhibitors such as imatinib have a great therapeutic potential because of the many signaling events that protein kinases mediate. However, these drugs have to be exceptionally specific for their target kinase and resistance mutations can render these drugs ineffective. The proposed experiments will clarify the interplay between kinase dynamics, drug specificity, drug resistance mutations and kinase regulation.
|Foda, Zachariah H; Shan, Yibing; Kim, Eric T et al. (2015) A dynamically coupled allosteric network underlies binding cooperativity in Src kinase. Nat Commun 6:5939|
|Maianti, Juan Pablo; McFedries, Amanda; Foda, Zachariah H et al. (2014) Anti-diabetic activity of insulin-degrading enzyme inhibitors mediated by multiple hormones. Nature 511:94-8|
|Georghiou, George; Kleiner, Ralph E; Pulkoski-Gross, Michael et al. (2012) Highly specific, bisubstrate-competitive Src inhibitors from DNA-templated macrocycles. Nat Chem Biol 8:366-74|
|Montpetit, Ben; Seeliger, Markus A; Weis, Karsten (2012) Analysis of DEAD-box proteins in mRNA export. Methods Enzymol 511:239-54|
|Wang, Likun; Perera, B Gayani K; Hari, Sanjay B et al. (2012) Divergent allosteric control of the IRE1ýý endoribonuclease using kinase inhibitors. Nat Chem Biol 8:982-9|
|Jura, Natalia; Zhang, Xuewu; Endres, Nicholas F et al. (2011) Catalytic control in the EGF receptor and its connection to general kinase regulatory mechanisms. Mol Cell 42:9-22|
|Montpetit, Ben; Thomsen, Nathan D; Helmke, Kara J et al. (2011) A conserved mechanism of DEAD-box ATPase activation by nucleoporins and InsP6 in mRNA export. Nature 472:238-42|
|Shan, Yibing; Kim, Eric T; Eastwood, Michael P et al. (2011) How does a drug molecule find its target binding site? J Am Chem Soc 133:9181-3|
|Weisberg, Ellen; Choi, Hwan Geun; Ray, Arghya et al. (2010) Discovery of a small-molecule type II inhibitor of wild-type and gatekeeper mutants of BCR-ABL, PDGFRalpha, Kit, and Src kinases: novel type II inhibitor of gatekeeper mutants. Blood 115:4206-16|