Protein Kinase Inhibitors in Research and Medicine

1. Auflage 2013
ISBN: 978-0-12-398462-3
Verlag: Elsevier Science & Techn.
Format: EPUB
Kopierschutz: 6 - ePub Watermark

E-Book, Englisch, Band Volume 548, 252 Seiten

Reihe: Methods in Enzymology

This new volume of Methods in Enzymology continues the legacy of this premier serial with quality chapters authored by leaders in the field. This volume covers protein kinase inhibitors in research and medicine, and includes chapters on such topics as fragment-based screening, broad kinome profiling of kinase inhibitors, and designing drug-resistant kinase alleles. - Continues the legacy of this premier serial with quality chapters authored by leaders in the field - Covers research methods in biomineralization science - Contains sections focusing on protein kinase inhibitors in research and medicine

Autoren/Hrsg.

Inhaltsverzeichnis

1;Front Cover;1
2;Protein Kinase Inhibitors in Research and Medicine ;4
3;Copyright;5
4;Contents;6
5;Contributors;10
6;Preface;12
7;Chapter One: Catalytic Mechanisms and Regulation of Protein Kinases;14
7.1;1. Introduction;14
7.2;2. Kinetic Mechanism;15
7.3;3. Chemical Mechanism of Kinase Phosphoryl Transfer;17
7.4;4. Applications of Mechanistic Studies in Understanding Kinase Function and Regulation;22
7.4.1;4.1. Bisubstrate analogs;22
7.4.2;4.2. Oncogenic kinase mutants;23
7.4.3;4.3. Chemical rescue of tyrosine kinases;27
7.5;5. Summary and Outlook;29
7.6;References;30
8;Chapter Two: A Structural Atlas of Kinases Inhibited by Clinically Approved Drugs;36
8.1;1. Introduction;37
8.2;2. Kinase Structure and Catalytic Mechanism;39
8.3;3. Staurosporine: A Promiscuous ATP-Competitive Inhibitor;44
8.4;4. BCR-Abl Inhibitors;46
8.4.1;4.1. Imatinib binds to a ``DFG-out´´ Abl conformation;46
8.4.2;4.2. Nilotinib (Tasigna): An imatinib analog effective against several imatinib-resistant Abl variants;49
8.4.3;4.3. Ponatinib (Iclusig) overcomes an Abl gatekeeper resistance mutation;50
8.4.4;4.4. Bosutinib (Bosulif) inhibits BCR-Abl;52
8.4.5;4.5. Dasatinib (Sprycel) binds to a ``DFG-in´´ conformation of Abl;52
8.5;5. Tofacitinib (Xeljanz) Binds to a ``DFG-in´´ Conformation of Janus Kinase;54
8.6;6. Inhibition of Receptor Tyrosine Kinases;55
8.6.1;6.1. Imatinib binds to a ``DFG-out´´ conformation of c-Kit;55
8.6.2;6.2. Inhibitors of vascular endothelial growth factor receptor;57
8.6.3;6.3. Crizotinib (Xalkori) binds a ``DFG-in´´ conformation of ALK and c-MET;60
8.6.4;6.4. Gefitinib (Iressa) and erlotinib (Tarceva) inhibit EGFR;61
8.6.5;6.5. Development of a more selective EGFR inhibitor: Lapatinib (Tykerb);61
8.6.6;6.6. Afatinib (Gilotrif): A selective kinase inhibitor that reacts covalently;64
8.6.7;6.7. Vandetanib (Caprelsa): A quinazoline analog that inhibits RET;65
8.7;7. Vemurafenib (Zelboraf) Binds to A ``DFG-in´´ Conformation in the Ser/Thr Kinase RAF;66
8.8;8. Inhibitors That Occupy Pockets Other Than the ATP-Binding Site;68
8.8.1;8.1. Benzothiazines bind an allosteric site in focal adhesion kinase;68
8.8.2;8.2. PD318088 binds to MEK noncompetitively with ATP;68
8.8.3;8.3. Inhibitors that bind to the kinase domain to disrupt substrate recruitment;71
8.9;9. Summary;71
8.10;Acknowledgments;71
8.11;References;72
9;Chapter Three: Fragment-Based Approaches to the Discovery of Kinase Inhibitors;82
9.1;1. Introduction;83
9.1.1;1.1. Challenges of kinases as drug targets;84
9.2;2. Fragment-Based Drug Discovery;85
9.2.1;2.1. Advantages of FBDD;87
9.2.2;2.2. Challenges of FBDD;88
9.2.3;2.3. Discovering kinase inhibitors with FBDD;89
9.2.4;2.4. FBDD-derived kinase inhibitors in the clinic;89
9.3;3. Identifying Fragment Hits;91
9.3.1;3.1. Library construction;91
9.3.2;3.2. Hit identification;91
9.3.3;3.3. Hit validation;92
9.4;4. From Fragments to Leads;93
9.4.1;4.1. Selection of hits for optimization;93
9.4.2;4.2. Structure-guided optimization;95
9.4.3;4.3. Achieving selective inhibition;95
9.5;5. Alternative Inhibition Strategies;96
9.5.1;5.1. Type II inhibition;97
9.5.2;5.2. Type III inhibition;97
9.5.3;5.3. Other modes of inhibition;98
9.6;6. Summary;99
9.7;Acknowledgments;101
9.8;References;101
10;Chapter Four: Targeting Protein Kinases with Selective and Semipromiscuous Covalent Inhibitors;106
10.1;1. Introduction;107
10.2;2. Design of Irreversible Cysteine-Targeted Kinase Inhibitors;108
10.2.1;2.1. Irreversible covalent inhibitors of RSK1/2/4;109
10.2.1.1;2.1.1. Applications of irreversible covalent kinase inhibitors;111
10.2.2;2.2. Fluorescent and alkyne-tagged probes to quantify proteome-wide selectivity and RSK occupancy in vivo;112
10.2.2.1;2.2.1. Assessing RSK1/2 occupancy after dosing mice with FMK-MEA;115
10.3;3. Targeting Noncatalytic Cysteines with Reversible Covalent Inhibitors;117
10.3.1;3.1. Reversible Michael acceptors for cysteine-targeting applications;117
10.3.2;3.2. Electrophilic fragment-based ligand discovery with cyanoacrylamides;118
10.3.2.1;3.2.1. Assembling and screening a cyanoacrylamide fragment library;120
10.4;4. Semipromiscuous Covalent Inhibitors as Chemoproteomic Probes;122
10.4.1;4.1. Identification of new therapeutic kinase targets with a semipromiscuous inhibitor;122
10.4.2;4.2. Targeting the catalytic lysine with covalent probes;123
10.5;5. Conclusions and Future Directions;126
10.6;References;127
11;Chapter Five: The Resistance Tetrad: Amino Acid Hotspots for Kinome-Wide Exploitation of Drug-Resistant Protein Kinase Al...;130
11.1;1. Introduction;131
11.2;2. Protein Kinases and Kinase Inhibitors;132
11.3;3. Protein Kinase Inhibitors;132
11.4;4. Screening Approaches to Decipher Protein Kinase-Inhibitor Specificity;135
11.5;5. Random and Directed Mutagenesis Approaches Reveal Common Resistance Mechanisms;137
11.6;6. A General Procedure for Directed (Nonrandom) Mutagenesis of dsDNA Plasmids;139
11.7;7. The Resistance Tetrad Position 0: The Gatekeeper Residue;140
11.8;8. SB203580: A Paradigm for Gatekeeper-Mediated Drug Resistance from Test Tube to Mouse;142
11.9;9. Expanding the Resistance Tetrad: +2 (Hydrophobic) and +6/+7 Specificity Surfaces in Kinases;143
11.10;10. The Resistance Tetrad is a Selectivity Filter Applicable for Kinome-Wide Drug-Resistance Studies;144
11.11;11. Engineering and Analysis of Logically Designed Drug-Resistance Mutations;145
11.12;12. Analysis of Inhibitor Resistance Toward WT and DR Mutants In Vitro;147
11.13;13. Oncogenic Gatekeeper Mutations: Unanticipated Mechanisms of Gatekeeper Resistance Merit Biochemical Scrutiny of DR Mu...;148
11.14;14. Evaluation of Catalytic Behavior and KM[ATP] Value for WT and DR Kinase Mutants In Vitro;149
11.15;15. Intact Cell Systems for Analyzing Drug Resistance and Target Validation;150
11.16;16. Generation of Stable, Isogenic Cell Lines Expressing Tetracycline-Inducible Kinases;150
11.16.1;16.1. Transfection and selection procedure;151
11.17;17. Analysis of Kinase Drug Resistance Toward a Cytotoxic Inhibitor: Cell Growth Assay Based on Colony Formation (Fig.5.2F);152
11.18;18. Conclusions;152
11.19;References;153
12;Chapter Six: FLiK: A Direct-Binding Assay for the Identification and Kinetic Characterization of Stabilizers of Inactive ...;160
12.1;1. Introduction;161
12.2;2. Design and Preparation of Kinases for FLiK;165
12.2.1;2.1. Selection of the labeling position on the activation loop;165
12.2.2;2.2. Preparation of p38a MAP kinase construct for FLiK;166
12.2.2.1;2.2.1. Expression of p38a MAP kinase;167
12.2.2.2;2.2.2. Purification of p38a MAP kinase;167
12.3;3. Labeling of p38a MAP Kinase with Acrylodan;168
12.4;4. Assay Characterization and Validation;169
12.4.1;4.1. Measure emission spectra for each kinase conformation;170
12.4.2;4.2. Kd determination;172
12.4.2.1;4.2.1. Titration of ligand with the FLiK kinase (for rapidly binding ligands);173
12.4.2.2;4.2.2. Titration of ligand with the FLiK kinase (for slow-binding ligands);174
12.4.3;4.3. Kinetic measurements;174
12.4.3.1;4.3.1. Determination of kon;175
12.4.3.2;4.3.2. Determination of koff;177
12.5;5. HTS with FLiK;177
12.5.1;5.1. Adaptation to HTS formats;177
12.5.2;5.2. HTS of compound libraries;179
12.5.3;5.3. Data analysis, fluorescence artifacts, and pitfalls;180
12.6;6. Summary;182
12.7;Acknowledgments;183
12.8;References;183
13;Chapter Seven: Discovery of Allosteric Bcr-Abl Inhibitors from Phenotypic Screen to Clinical Candidate;186
13.1;1. Development of ATP-Site-Directed Inhibitors of BCR-ABL for the Treatment of CML;187
13.2;2. Discovery and Characterization of Non-ATP-Site-Directed BCR-ABL Inhibitors;189
13.3;3. Characterization of the Binding of the Non-ATP-Site-Directed Bcr-ABL Inhibitor GNF-2;192
13.4;4. Therapeutic Potential of First-Generation myr-Pocket Binders;196
13.4.1;4.1. Single-agent activity;196
13.4.2;4.2. Combinations with ATP-competitive ligands;197
13.4.3;4.3. Second-generation myr-pocket binders;197
13.5;5. Combinations of Second-Generation ATP-Site Inhibitors with Second-Generation myr-Pocket Ligands;198
13.5.1;5.1. Key lessons learned in the drug discovery of allosteric BCR-ABL inhibitors;198
13.6;Acknowledgments;199
13.7;References;199
14;Chapter Eight: The Logic and Design of Analog-Sensitive Kinases and Their Small Molecule Inhibitors;202
14.1;1. Introduction;203
14.1.1;1.1. Overview of analog-sensitive-kinase technology;203
14.1.2;1.2. The gatekeeper governs access to the ATP-binding pocket of protein kinases;204
14.2;2. Constructing AS Kinases;206
14.2.1;2.1. Identifying the gatekeeper residue;206
14.2.2;2.2. Second-site suppressor mutations;208
14.2.3;2.3. Unnatural ATP analogs rescue enzyme activity;209
14.2.4;2.4. Cysteine gatekeeper alternative;209
14.3;3. AS Kinase Inhibitors;210
14.3.1;3.1. Pyrazolo[3,4-d]pyrimidine inhibitors;211
14.3.2;3.2. Synthesis of PP inhibitors;212
14.3.3;3.3. Staralog inhibitors;212
14.3.4;3.4. Synthesis of staralog inhibitors;213
14.3.5;3.5. ES-kinase inhibitors;216
14.3.6;3.6. Synthesis of ES-kinase PP inhibitors;216
14.3.7;3.7. AS Kinase inhibitors for PI3-like kinases;217
14.3.8;3.8. Protocol for measuring inhibitor potency;217
14.4;4. AS Kinases in Cells;220
14.4.1;4.1. AS Kinases in yeast;221
14.4.2;4.2. AS Kinases in mammalian cells;221
14.5;5. AS Kinases in Living Multicellular Organisms;222
14.6;6. Summary;223
14.7;References;224
15;Author Index;228
16;Subject Index;248
17;Color Plate;255

Leseproben

Chapter One Catalytic Mechanisms and Regulation of Protein Kinases
Zhihong Wang*; Philip A. Cole†,1    * Department of Chemistry and Biochemistry, University of the Sciences, Philadelphia, Pennsylvania, USA
† Department of Pharmacology and Molecular Sciences, Johns Hopkins School of Medicine, Baltimore, Maryland, USA
1 Corresponding author: email address: [e-mail] Abstract
Protein kinases transfer a phosphoryl group from ATP onto target proteins and play a critical role in signal transduction and other cellular processes. Here, we review the kinase kinetic and chemical mechanisms and their application in understanding kinase structure and function. Aberrant kinase activity has been implicated in many human diseases, in particular cancer. We highlight applications of technologies and concepts derived from kinase mechanistic studies that have helped illuminate how kinases are regulated and contribute to pathophysiology. Keywords Transition state Bisubstrate analog Inhibitor Erlotinib Lapatinib B-Raf EGFR Src Chemical rescue 1 Introduction
The discovery of protein kinases in the 1950s led to a massive influence on clarifying biological pathways and disease mechanisms and developing therapies over the subsequent six decades (Hunter, 2000; Krebs & Beavo, 1979). Eukaryotic protein kinases are enzymes that catalyze phosphoryl transfer from MgATP to Ser/Thr and Tyr side chains in proteins. Their importance is in part evidenced by their frequency in eukaryotic genomes, typically representing 2–3% of the genes, including in human where 518 protein kinases have been annotated (Manning, Whyte, Martinez, Hunter, & Sudarsanam, 2002). While each specific kinase is thought to have a specialized function, there are many conserved features among kinases regarding their structures and catalytic mechanisms (Hanks, Quinn, & Hunter, 1988). This protein kinase chapter is written from an enzymology perspective and will cover the kinetic and chemical mechanisms of kinases and how an understanding of these features has been used to explore the structure, function, and regulation of these important catalysts. 2 Kinetic Mechanism
Protein kinases operate on two substrates, proteins, and MgATP and produce phosphoproteins and MgADP (Adams, 2001; Taylor & Kornev, 2011). While it is sometimes the case that free ATP rather than Mg-bound ATP is thought of as the phosphoryl-donor substrate, the affinity of Mg for ATP is high enough that there is only a low concentration of non-Mg-bound ATP in cells. Thus with one apparent exception (Mukherjee et al., 2008), protein kinases require at least one divalent ion, Mg or Mn, for catalysis. Two substrate group transfer enzymes like kinases can be classified into two general types, those that follow ternary complex mechanisms and those that follow ping-pong mechanisms (Segel, 1993). Ternary complex mechanisms typically involve direct reaction between the two substrates to afford the two products, whereas ping-pong mechanisms proceed through a covalent enzyme intermediate, which in the case of kinases would be a phosphoenzyme species. Classical two substrate steady-state kinetics experiments revealing an intersecting line pattern in double reciprocal plots (Segel, 1993) as well as more technically sophisticated stereochemical studies showing inversion at the phosphoryl group (Knowles, 1980) helped define protein kinase A (PKA) as following a ternary complex mechanism. Subsequently, two substrate kinetic studies on a variety of Ser/Thr and Tyr kinases and many X-ray structures of these enzymes in complex with substrate analogs have confirmed this to be a general feature of the kinase superfamily (Zheng et al., 1993). However, recently, an X-ray crystal structure of an atypical kinase showed the surprising finding that an active site aspartate was phosphorylated (Ferreira-Cerca et al., 2012). This phosphoAsp was proposed to correspond to a phosphoenzyme intermediate that could deliver the phosphoryl group to a protein substrate, though further experiments will be needed to establish this mechanism. Of note, nucleoside diphosphokinase does proceed through a phosphohistidine intermediate so there is enzymatic precedence for a small-molecule kinase using a related mechanism (Admiraal et al., 1999). For the vast majority of protein kinases that involve direct phosphoryl transfer through a ternary complex, other kinetic mechanism issues that have been addressed are whether there is a preference for MgATP or protein substrate to bind first and what step(s) is rate-limiting for catalysis? These features have been analyzed for a variety of protein kinases and the results are somewhat enzyme and reaction condition dependent. For example, PKA displays a clear preference for MgATP binding prior to peptide substrate whereas Csk kinase shows no apparent-binding preference between nucleotide or peptide substrates (Cole, Burn, Takacs, & Walsh, 1994; Qamar, Yoon, & Cook, 1992; Zheng et al., 1993). Interestingly, experiments on p38 MAP kinase have led to contradictory models. Models in which protein substrate binds first, MgATP binds first, or random order binding have all been proposed for p38 MAP kinase (LoGrasso et al., 1997; Szafranska & Dalby, 2005). While these different models could be traced to the distinct methods used for measurement, they also highlight the limitation of steady-state kinetic approaches to provide unambiguous mechanistic portraits. The most comprehensive studies on p38 MAP kinase that include complementary methods including calorimetry and structural considerations point to a random order of substrate binding for this enzyme (Szafranska & Dalby, 2005). One potential practical application that emanates from such models relates to the development of specific kinase inhibitors that target the ATP pocket (Noble, Endicott, & Johnson, 2004). If the protein substrate binds to the kinase in the absence of MgATP, there may be an influence on drug affinity. Regarding rate-limiting steps, a combination of viscosity effects, presteady-state kinetic techniques, and alternate substrates have been employed with various kinases to define the microscopic rate constants. To some extent, the kinetic models not only depend on the conditions of the kinase assay conditions (salt concentration, divalent ion (Mg vs. Mn), peptide, or protein substrate), but they also show differences among the kinases themselves. With PKA, MgADP release is fully rate determining (Adams & Taylor, 1992; Qamar & Cook, 1993), whereas for Csk phosphoryl transfer is partially or fully rate limiting depending on whether MgATP or MnATP is used as the substrate (Grace, Walsh, & Cole, 1997). When Mg is used with Csk, product release is fast and chemistry is rate determining, whereas when Mn is employed, product release slows down, presumably because of metal–enzyme interactions. Increasing the ionic strength of the buffer can also speed product release, possibly by weakening the interactions between nucleotide and enzyme. Some kinases such as Ser–Arg protein kinase or Src protein tyrosine kinase can show processive phosphorylation of its protein substrate, effectively indicating that protein substrate/product release is the slow step in turnover (Aubol et al., 2003; Pellicena & Miller, 2001). Furthermore, many protein kinases like the insulin receptor tyrosine kinase (IRK) are regulated by accessory domains, phosphorylation, or allosteric ligands which can dramatically impact the nature of the rate-limiting steps (Ablooglu, Frankel, Rusinova, Ross, & Kohanski, 2001; Hubbard & Miller, 2007). 3 Chemical Mechanism of Kinase Phosphoryl Transfer
Despite the apparent simplicity of the reaction chemistry, there has been significant effort to understand the details of how the phosphoryl group moves from ATP to the protein substrate hydroxy group in the kinase active site. This interest stems from several considerations. One is the fundamental challenge in defining the catalytic mechanism of an important family of enzymes. A second factor relates to our fascination with how kinase enzymes interconvert between more active and less active forms. Kinase regulation by ligands, phosphorylation, as well as mutation can alter the alignment of active site residues, which ultimately translates to effects on the chemistry of phosphoryl transfer. A third reason for interest in the chemical mechanism is to aid in the design of synthetic compounds which can artificially switch kinase activity on or off. Such mechanism-inspired chemical biology approaches can and have shed light on the biological functions of kinases in cellular signaling. A central issue in defining the kinase mechanism is clarifying the nature of the phosphoryl transfer transition state. In the study of nonenzymatic phosphoryl transfer mechanisms, research dating back to the 1960s showed that phosphate monoesters like phenol phosphates display “dissociative” transition states (Kirby & Jencks, 1965). A dissociative transition state is one in which the bond between the phosphorus atom and the leaving group is largely broken prior to significant bond formation...