Protein Kinase Inhibitors in Research and Medicine (eBook)

Kevan M Shokat (Herausgeber)

eBook Download: PDF | EPUB

2013 | 1. Auflage
252 Seiten
Elsevier Science (Verlag)
978-0-12-398462-3 (ISBN)

Infos

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

Inhalt

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

Textauszug

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...

Produkt-Details

• Erscheint lt. Verlag: 1.7.2013
• Sprache: englisch
• Themenwelt: Medizin / Pharmazie ► Medizinische Fachgebiete ► Pharmakologie / Pharmakotherapie
• Themenwelt: Naturwissenschaften ► Biologie ► Biochemie
• Themenwelt: Naturwissenschaften ► Physik / Astronomie ► Angewandte Physik
• ISBN-10: 0-12-398462-9 / 0123984629
• ISBN-13: 978-0-12-398462-3 / 9780123984623