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.