Kinases are the second-largest drug-target family with 10 approved kinase inhibitor drugs and 50 compounds in clinical trials. Protein-kinase-domains are most frequently encoded by cancer-genes. Several cancer-driving mutations occur in their ATP-binding G-loops. The Abl-inhibitor Imatinib is a breakthrough-therapeutic for chronic-myelogenous-leukemia, but ~35% of the patients relapse due to accumulation of Imatinib-resistant Abl kinase-domain-mutations, particularly in the G-loop. Drug-resistance could thus become a major clincial problem as increasing patient populations are treated with kinase-inhibitor drugs. Using the Src-family protein tyrosine kinase Lyn as an experimentally very tractable example, we propose to implement and validate a multidisciplinary approach that first uses molecular dynamics (MD) simulations to relatively quickly identify mutations that affect catalysis and inhibitor interactions and can cause drug-resistance (Aim 1). Our approach next analyzes the activities, inhibitor-interactions and -resistance of the identified Lyn mutants in vitro and in vivo in Ba/F3 cells (Aim 2) or in Lyn-/- bone-marrow (Aim 3) to identify those mutations that are most relevant physiologically. Exclusion of uninformative mutants at each step minimizes experimental effort and maximizes relevance and likelihood of success. We consider this integrated approach to discover drug-resistance causing kinase mutations highly innovative, because it provides important insight that is usually only gained over much longer time periods and through the efforts of several labs. These studies follow up on our recently published finding that 58 eukaryotic kinases contain a conserved electrostatic salt-bridge across their G-loops that is essential for G-loop-stabilization, catalysis and ATP- or ATP-competitive inhibitor-binding. Salt-bridge- disruption in Bcr-Abl causes Imatinib-resistance. Our preliminary data suggest that in 31 kinases, including the Src, Abl, CK1 and CK2-families which all have important roles in cancer, the acidic salt-bridge-anchor also interacts electrostatically with a conserved polar-aromatic or basic amino-acid-side-chain embedded in a hydrophobic core. To test the hypothesis that this "triad interaction-network" architecture is essential for G-loop function and inhibitor-interactions, and that its disruption can cause drug resistance, we will analyze the effects of mutationally modulating the different components of the variant G-loop-triad-configurations in the exemplary kinases Lyn (Aims 1-3), Abl, CK1(2 and CK2a1 (Aim 4). To keep Aim 4 achievable within the 5 year funding period, we will focus on MD analyses. Future research will analyze the predicted high-priority mutants in vitro and in vivo. We consider this proposal highly significant, because it implements and validates an efficient approach to understand the molecular mechanisms through which a therapeutically very important target class functions, interacts with small-molecule inhibitors and can become drug-resistant. If successful, our approach can be applied to other targets to identify drug-resistant mutants at the onset of a drug discovery project, enabling the structure-based rational design of molecules that inhibit wildtype and mutant kinases potently. This will aid the development of more selective, less side-effect and less drug-resistance prone therapeutics.
While kinases have become the second-largest family of drug targets due to their paramount roles in causing diseases such as cancer, the accumulation of drug-resistant mutant kinases in patients treated with kinase inhibitor drugs remains a major therapeutic problem. To promote our understanding of the molecular mechanisms mediating kinase function, small-molecule inhibition and in particular the development of drug- resistance, we propose an innovative, multidisciplinary approach that, if successful, can be applied to any potential drug-target protein whose structure is known. The results will advance our understanding of kinase- function and aid the development of new and improved kinase-inhibitor drugs that are less prone to undesired side-effects, and less susceptible to drug-resistance. .
|Siegemund, Sabine; Shepherd, Jovan; Xiao, Changchun et al. (2015) hCD2-iCre and Vav-iCre mediated gene recombination patterns in murine hematopoietic cells. PLoS One 10:e0124661|
|Siegemund, Sabine; Rigaud, Stephanie; Conche, Claire et al. (2015) IP3 3-kinase B controls hematopoietic stem cell homeostasis and prevents lethal hematopoietic failure in mice. Blood 125:2786-97|
|Sauer, Karsten; Okkenhaug, Klaus (2015) Editorial: Lipid Signaling in T Cell Development and Function. Front Immunol 6:410|
|Xu, Xiaolu; Jaeger, Emily R; Wang, Xinxin et al. (2014) Mst1 directs Myosin IIa partitioning of low and higher affinity integrins during T cell migration. PLoS One 9:e105561|
|Mukherjee, Sayak; Rigaud, Stephanie; Seok, Sang-Cheol et al. (2014) Correction: In Silico Modeling of Itk Activation Kinetics in Thymocytes Suggests Competing Positive and Negative IP4 Mediated Feedbacks Increase Robustness. PLoS One 9:|
|Mukherjee, Sayak; Rigaud, Stephanie; Seok, Sang-Cheol et al. (2013) In silico modeling of Itk activation kinetics in thymocytes suggests competing positive and negative IP4 mediated feedbacks increase robustness. PLoS One 8:e73937|
|Sauer, Karsten; Park, Eugene; Siegemund, Sabine et al. (2013) Inositol tetrakisphosphate limits NK cell effector functions by controlling PI3K signaling. Blood 121:286-97|
|Fu, Guo; Casas, Javier; Rigaud, Stephanie et al. (2013) Themis sets the signal threshold for positive and negative selection in T-cell development. Nature 504:441-5|
|Zhao, Yanxiang; Kwan, Yuen-Yick; Che, Jianwei et al. (2013) Phase-field approach to implicit solvation of biomolecules with Coulomb-field approximation. J Chem Phys 139:024111|
|Guo, Zuojun; Li, Bo; Dzubiella, Joachim et al. (2013) Evaluation of Hydration Free Energy by Level-Set Variational Implicit-Solvent Model with Coulomb-Field Approximation. J Chem Theory Comput 9:1778-1787|
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