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.

Public Health Relevance

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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Transition Award (R00)
Project #
5R00GM080097-05
Application #
8197751
Study Section
Special Emphasis Panel (NSS)
Program Officer
Wehrle, Janna P
Project Start
2007-07-15
Project End
2013-11-30
Budget Start
2011-12-01
Budget End
2013-11-30
Support Year
5
Fiscal Year
2012
Total Cost
$246,510
Indirect Cost
$75,120
Name
State University New York Stony Brook
Department
Pharmacology
Type
Schools of Medicine
DUNS #
804878247
City
Stony Brook
State
NY
Country
United States
Zip Code
11794
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
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
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