Note: Front Cover -- Protein Kinase Inhibitors in Research and Medicine -- Copyright -- Contents -- Contributors -- Preface -- Chapter One: Catalytic Mechanisms and Regulation of Protein Kinases -- 1. Introduction -- 2. Kinetic Mechanism -- 3. Chemical Mechanism of Kinase Phosphoryl Transfer -- 4. Applications of Mechanistic Studies in Understanding Kinase Function and Regulation -- 4.1. Bisubstrate analogs -- 4.2. Oncogenic kinase mutants -- 4.3. Chemical rescue of tyrosine kinases -- 5. Summary and Outlook -- References -- Chapter Two: A Structural Atlas of Kinases Inhibited by Clinically Approved Drugs -- 1. Introduction -- 2. Kinase Structure and Catalytic Mechanism -- 3. Staurosporine: A Promiscuous ATP-Competitive Inhibitor -- 4. BCR-Abl Inhibitors -- 4.1. Imatinib binds to a ``DFG-out´´ Abl conformation -- 4.2. Nilotinib (Tasigna): An imatinib analog effective against several imatinib-resistant Abl variants -- 4.3. Ponatinib (Iclusig) overcomes an Abl gatekeeper resistance mutation -- 4.4. Bosutinib (Bosulif) inhibits BCR-Abl -- 4.5. Dasatinib (Sprycel) binds to a ``DFG-in´´ conformation of Abl -- 5. Tofacitinib (Xeljanz) Binds to a ``DFG-in´´ Conformation of Janus Kinase -- 6. Inhibition of Receptor Tyrosine Kinases -- 6.1. Imatinib binds to a ``DFG-out´´ conformation of c-Kit -- 6.2. Inhibitors of vascular endothelial growth factor receptor -- 6.3. Crizotinib (Xalkori) binds a ``DFG-in´´ conformation of ALK and c-MET -- 6.4. Gefitinib (Iressa) and erlotinib (Tarceva) inhibit EGFR -- 6.5. Development of a more selective EGFR inhibitor: Lapatinib (Tykerb) -- 6.6. Afatinib (Gilotrif): A selective kinase inhibitor that reacts covalently -- 6.7. Vandetanib (Caprelsa): A quinazoline analog that inhibits RET -- 7. Vemurafenib (Zelboraf) Binds to A ``DFG-in´´ Conformation in the Ser/Thr Kinase RAF. , 8. Inhibitors That Occupy Pockets Other Than the ATP-Binding Site -- 8.1. Benzothiazines bind an allosteric site in focal adhesion kinase -- 8.2. PD318088 binds to MEK noncompetitively with ATP -- 8.3. Inhibitors that bind to the kinase domain to disrupt substrate recruitment -- 9. Summary -- Acknowledgments -- References -- Chapter Three: Fragment-Based Approaches to the Discovery of Kinase Inhibitors -- 1. Introduction -- 1.1. Challenges of kinases as drug targets -- 2. Fragment-Based Drug Discovery -- 2.1. Advantages of FBDD -- 2.2. Challenges of FBDD -- 2.3. Discovering kinase inhibitors with FBDD -- 2.4. FBDD-derived kinase inhibitors in the clinic -- 3. Identifying Fragment Hits -- 3.1. Library construction -- 3.2. Hit identification -- 3.3. Hit validation -- 4. From Fragments to Leads -- 4.1. Selection of hits for optimization -- 4.2. Structure-guided optimization -- 4.3. Achieving selective inhibition -- 5. Alternative Inhibition Strategies -- 5.1. Type II inhibition -- 5.2. Type III inhibition -- 5.3. Other modes of inhibition -- 6. Summary -- Acknowledgments -- References -- Chapter Four: Targeting Protein Kinases with Selective and Semipromiscuous Covalent Inhibitors -- 1. Introduction -- 2. Design of Irreversible Cysteine-Targeted Kinase Inhibitors -- 2.1. Irreversible covalent inhibitors of RSK1/2/4 -- 2.1.1. Applications of irreversible covalent kinase inhibitors -- 2.2. Fluorescent and alkyne-tagged probes to quantify proteome-wide selectivity and RSK occupancy in vivo -- 2.2.1. Assessing RSK1/2 occupancy after dosing mice with FMK-MEA -- 3. Targeting Noncatalytic Cysteines with Reversible Covalent Inhibitors -- 3.1. Reversible Michael acceptors for cysteine-targeting applications -- 3.2. Electrophilic fragment-based ligand discovery with cyanoacrylamides -- 3.2.1. Assembling and screening a cyanoacrylamide fragment library. , 4. Semipromiscuous Covalent Inhibitors as Chemoproteomic Probes -- 4.1. Identification of new therapeutic kinase targets with a semipromiscuous inhibitor -- 4.2. Targeting the catalytic lysine with covalent probes -- 5. Conclusions and Future Directions -- References -- Chapter Five: The Resistance Tetrad: Amino Acid Hotspots for Kinome-Wide Exploitation of Drug-Resistant Protein Kinase Al... -- 1. Introduction -- 2. Protein Kinases and Kinase Inhibitors -- 3. Protein Kinase Inhibitors -- 4. Screening Approaches to Decipher Protein Kinase-Inhibitor Specificity -- 5. Random and Directed Mutagenesis Approaches Reveal Common Resistance Mechanisms -- 6. A General Procedure for Directed (Nonrandom) Mutagenesis of dsDNA Plasmids -- 7. The Resistance Tetrad Position 0: The Gatekeeper Residue -- 8. SB203580: A Paradigm for Gatekeeper-Mediated Drug Resistance from Test Tube to Mouse -- 9. Expanding the Resistance Tetrad: +2 (Hydrophobic) and +6/+7 Specificity Surfaces in Kinases -- 10. The Resistance Tetrad is a Selectivity Filter Applicable for Kinome-Wide Drug-Resistance Studies -- 11. Engineering and Analysis of Logically Designed Drug-Resistance Mutations -- 12. Analysis of Inhibitor Resistance Toward WT and DR Mutants In Vitro -- 13. Oncogenic Gatekeeper Mutations: Unanticipated Mechanisms of Gatekeeper Resistance Merit Biochemical Scrutiny of DR Mu... -- 14. Evaluation of Catalytic Behavior and KM[ATP] Value for WT and DR Kinase Mutants In Vitro -- 15. Intact Cell Systems for Analyzing Drug Resistance and Target Validation -- 16. Generation of Stable, Isogenic Cell Lines Expressing Tetracycline-Inducible Kinases -- 16.1. Transfection and selection procedure -- 17. Analysis of Kinase Drug Resistance Toward a Cytotoxic Inhibitor: Cell Growth Assay Based on Colony Formation (Fig.5.2F) -- 18. Conclusions -- References. , Chapter Six: FLiK: A Direct-Binding Assay for the Identification and Kinetic Characterization of Stabilizers of Inactive ... -- 1. Introduction -- 2. Design and Preparation of Kinases for FLiK -- 2.1. Selection of the labeling position on the activation loop -- 2.2. Preparation of p38α MAP kinase construct for FLiK -- 2.2.1. Expression of p38α MAP kinase -- 2.2.2. Purification of p38α MAP kinase -- 3. Labeling of p38α MAP Kinase with Acrylodan -- 4. Assay Characterization and Validation -- 4.1. Measure emission spectra for each kinase conformation -- 4.2. Kd determination -- 4.2.1. Titration of ligand with the FLiK kinase (for rapidly binding ligands) -- 4.2.2. Titration of ligand with the FLiK kinase (for slow-binding ligands) -- 4.3. Kinetic measurements -- 4.3.1. Determination of kon -- 4.3.2. Determination of koff -- 5. HTS with FLiK -- 5.1. Adaptation to HTS formats -- 5.2. HTS of compound libraries -- 5.3. Data analysis, fluorescence artifacts, and pitfalls -- 6. Summary -- Acknowledgments -- References -- Chapter Seven: Discovery of Allosteric Bcr-Abl Inhibitors from Phenotypic Screen to Clinical Candidate -- 1. Development of ATP-Site-Directed Inhibitors of BCR-ABL for the Treatment of CML -- 2. Discovery and Characterization of Non-ATP-Site-Directed BCR-ABL Inhibitors -- 3. Characterization of the Binding of the Non-ATP-Site-Directed Bcr-ABL Inhibitor GNF-2 -- 4. Therapeutic Potential of First-Generation myr-Pocket Binders -- 4.1. Single-agent activity -- 4.2. Combinations with ATP-competitive ligands -- 4.3. Second-generation myr-pocket binders -- 5. Combinations of Second-Generation ATP-Site Inhibitors with Second-Generation myr-Pocket Ligands -- 5.1. Key lessons learned in the drug discovery of allosteric BCR-ABL inhibitors -- Acknowledgments -- References. , Chapter Eight: The Logic and Design of Analog-Sensitive Kinases and Their Small Molecule Inhibitors -- 1. Introduction -- 1.1. Overview of analog-sensitive-kinase technology -- 1.2. The gatekeeper governs access to the ATP-binding pocket of protein kinases -- 2. Constructing AS Kinases -- 2.1. Identifying the gatekeeper residue -- 2.2. Second-site suppressor mutations -- 2.3. Unnatural ATP analogs rescue enzyme activity -- 2.4. Cysteine gatekeeper alternative -- 3. AS Kinase Inhibitors -- 3.1. Pyrazolo[3,4-d]pyrimidine inhibitors -- 3.2. Synthesis of PP inhibitors -- 3.3. Staralog inhibitors -- 3.4. Synthesis of staralog inhibitors -- 3.5. ES-kinase inhibitors -- 3.6. Synthesis of ES-kinase PP inhibitors -- 3.7. AS Kinase inhibitors for PI3-like kinases -- 3.8. Protocol for measuring inhibitor potency -- 4. AS Kinases in Cells -- 4.1. AS Kinases in yeast -- 4.2. AS Kinases in mammalian cells -- 5. AS Kinases in Living Multicellular Organisms -- 6. Summary -- References -- Author Index -- Subject Index -- Color Plate.