Title Details

Part I Introduction – Epigenetics 1

1 Epigenetics:Moving Forward 3
Lucia Altucci

1.1 Why This Enormously Increased Interest? 4

1.2 Looking Forward to New Avenues of Epigenetics 5

Acknowledgments 7

References 7

Part II General Aspects/Methodologies 11

2 Structural Biology of Epigenetic Targets: Exploiting Complexity 13
Martin Marek, Tajith B. Shaik, and Christophe Romier

2.1 Introduction 13

2.2 DNA Methylases:The DNMT3A–DNMT3L–H3 and DNMT1–USP7 Complexes 14

2.3 Histone Arginine Methyltransferases:The PRMT5–MEP50 Complex 16

2.4 Histone Lysine Methyltransferases:The MLL3–RBBP5–ASH2L and the PRC2 Complexes 17

2.5 Histone Lysine Ubiquitinylases: The PRC1 Complex 21

2.6 Histone Lysine Deubiquitinylases: The SAGA Deubiquitination Module 22

2.7 Histone Acetyltransferases:The MSL1 and NUA4 Complexes 24

2.8 Histone Deacetylases: HDAC1–MTA1 and HDAC3–SMRT Complexes and HDAC6 26

2.9 Histone Variants and Histone Chaperones: A Complex and Modular Interplay 28

2.10 ATP-Dependent Remodelers: CHD1, ISWI, SNF2, and the SNF2-Nucleosome Complex 31

2.11 Epigenetic Readers: Histone Crotonylation Readers and the 53BP1-Nucleosome (H2AK15Ub–H4K20me2) Complex 35

2.12 Conclusions 37

Acknowledgments 38

References 38

3 Computer-based Lead Identification for Epigenetic Targets 45
Chiara Luise, Tino Heimburg, Berin Karaman, Dina Robaa, andWolfgang Sippl

3.1 Introduction 45

3.2 Computer-based Methods in Drug Discovery 46

3.2.1 Pharmacophore-based Methods 46

3.2.2 QSAR 47

3.2.3 Docking 47

3.2.4 Virtual Screening 48

3.2.5 Binding Free Energy Calculation 49

3.3 Histone Deacetylases 49

3.3.1 Zinc-Dependent HDACs 49

3.3.2 Sirtuins 54

3.4 Histone Methyltransferases 58

3.5 Histone Demethylases 61

3.5.1 LSD1 (KDM1A) 62

3.5.2 Jumonji Histone Demethylases 64

3.6 Summary 66

Acknowledgments 66

References 67

4 Mass Spectrometry and Chemical Biology in Epigenetics Drug Discovery 79
Christian Feller, DavidWeigt, and Carsten Hopf

4.1 Introduction: Mass Spectrometry Technology Used in Epigenetic Drug Discovery 79

4.1.1 Mass SpectrometryWorkflows for the Analysis of Proteins 80

4.1.2 Mass Spectrometry Imaging 83

4.2 Target Identification and Selectivity Profiling: Chemoproteomics 85

4.2.1 Histone Deacetylase and Acetyltransferase Chemoproteomics 87

4.2.2 Bromodomain Chemoproteomics 88

4.2.3 Demethylase Chemoproteomics 88

4.2.4 Methyltransferase Chemoproteomics 89

4.3 Characterization of Epigenetic Drug Target Complexes and Reader Complexes Contributing to Drug’s Mode of Action 89

4.3.1 Immunoaffinity Purification of Native Protein Complexes 89

4.3.2 Immunoaffinity Purification with Antibodies against Epitope Tags 90

4.3.3 Affinity Enrichment Using Histone Tail Peptides as Bait 91

4.4 Elucidation of a Drug’s Mode of Action: Analysis of Histone Posttranslational Modifications by MS-Based Proteomics 91

4.4.1 Histone Modification MS Workflows 92

4.4.2 Application of Histone MS Workflows to Characterize Epigenetic Drugs 95

4.5 Challenges and New Trends 97

4.5.1 Challenges and Trends in MS Analysis of Histone PTMs 97

4.5.2 High-Throughput Mass Spectrometry-Based Compound Profiling in Epigenetic Drug Discovery 98

4.5.3 Mass Spectrometry Imaging of Drug Action 98

Acknowledgments 99

References 99

5 PeptideMicroarrays for Epigenetic Targets 107
Alexandra Schutkowski, Diana Kalbas, Ulf Reimer, andMike Schutkowski

5.1 Introduction 107

5.2 Applications of Peptide Microarrays for Epigenetic Targets 110

5.2.1 Profiling of Substrate Specificities of Histone CodeWriters 110

5.2.2 Profiling of Substrate Specificities of Histone Code Erasers 114

5.2.3 Profiling of Binding Specificities of PTM-specific Antibodies and Histone Code Readers 117

5.2.3.1 Profiling of Specificities of PTM-specific Antibodies 118

5.2.3.2 Profiling of Binding Specificities of Histone Code Readers 119

5.2.4 Peptide Microarray-based Identification of Upstream Kinases and Phosphorylation Sites for Epigenetic Targets 121

5.3 Conclusion and Outlook 124

Acknowledgment 124

References 124

6 Chemical Probes 133
Amy Donner, Heather King, Paul E. Brennan, MosesMoustakim, andWilliam J. Zuercher

6.1 Chemical Probes Are Privileged Reagents for Biological Research 133

6.1.1 Best Practices for the Generation and Selection of Chemical Probes 134

6.1.2 Best Practices for Application of Chemical Probes 136

6.1.3 Cellular Target Engagement 137

6.1.3.1 Fluorescence Recovery after Photobleaching (FRAP) 138

6.1.3.2 Affinity Bead-Based Proteomics 138

6.1.3.3 Cellular Thermal Shift Assay (CETSA) 139

6.1.3.4 Bioluminescence Resonance Energy Transfer 139

6.2 Epigenetic Chemical Probes 141

6.2.1 Histone Acetylation and Bromodomain Chemical Probes 141

6.2.1.1 CBP/p300 Bromodomain Chemical Probes 144

6.2.1.2 Future Applications of Bromodomain Chemical Probes 147

6.3 Summary 147

References 148

Part III Epigenetic Target Classes 153

7 Inhibitors of the Zinc-Dependent Histone Deacetylases 155
Helle M. E. Kristensen, Andreas S. Madsen, and Christian A. Olsen

7.1 Introduction: Histone Deacetylases 155

7.2 Histone Deacetylase Inhibitors 158

7.2.1 Types of Inhibitors 158

7.2.2 HDAC Inhibitors in Clinical Use and Development 160

7.3 Targeting of HDAC Subclasses 169

7.3.1 Class I Inhibitors 169

7.3.1.1 HDAC1–3 Inhibitors 170

7.3.1.2 HDAC Inhibitors Targeting HDAC8 173

7.3.2 Class IIa Inhibitors 174

7.3.3 Class IIb 176

7.4 Perspectives 177

References 179

8 Sirtuins as Drug Targets 185
Clemens Zwergel, Dante Rotili, Sergio Valente, and Antonello Mai

8.1 Introduction 185

8.2 Biological Functions of Sirtuins in Physiology and Pathology 185

8.3 SIRT Modulators 188

8.3.1 SIRT Inhibitors 188

8.3.1.1 Small Molecules 188

8.3.1.2 Peptides and Pseudopeptides 191

8.3.2 SIRT Activators 191

8.4 Summary and Conclusions 192

References 193

9 Selective Small-Molecule Inhibitors of Protein Methyltransferases 201
H. Ümit Kaniskan and Jian Jin

9.1 Introduction 201

9.2 Protein Methylation 201

9.3 Lysine Methyltransferases (PKMTs) 202

9.4 Inhibitors of PKMTs 202

9.4.1 Inhibitors of H3K9 Methyltransferases 202

9.4.2 Inhibitors of H3K27 Methyltransferases 204

9.4.3 Inhibitors of H3K4 and H3K36 Methyltransferases 206

9.4.4 Inhibitors of H4K20 Methyltransferases 208

9.4.5 Inhibitors of H3K79 Methyltransferases 210

9.5 Protein Arginine Methyltransferases (PRMTs) 211

9.5.1 Inhibitors of PRMT1 211

9.5.2 Inhibitors of PRMT3 212

9.5.3 Inhibitors of CARM1 213

9.5.4 Inhibitors of PRMT5 214

9.5.5 Inhibitors of PRMT6 214

9.6 Concluding Remarks 215

References 215

10 LSD (Lysine-Specific Demethylase): A Decade-Long Trip from Discovery to Clinical Trials 221
Adam Lee, M. Teresa Borrello, and A. Ganesan

10.1 Introduction 221

10.2 LSDs: Discovery and Mechanistic Features 223

10.3 LSD Substrates 225

10.4 LSD Function and Dysfunction 229

10.5 LSD Inhibitors 232

10.5.1 Irreversible Small Molecule LSD Inhibitors from MAO Inhibitors 233

10.5.2 Reversible Small Molecule LSD Inhibitors 241

10.5.3 Synthetic Macromolecular LSD Inhibitors 248

10.6 Summary 251

References 253

11 JmjC-domain-Containing Histone Demethylases 263
Christoffer Højrup, Oliver D. Coleman, John-Paul Bukowski, Rasmus P. Clausen, and Akane Kawamura

11.1 Introduction 263

11.1.1 The LSD and JmjC Histone Lysine Demethylases 263

11.1.2 Histone Lysine Methylation and the JmjC-KDMs 265

11.1.3 The JmjC-KDMs in Development and Disease 266

11.2 KDM Inhibitor Development Targeting the JmjC Domain 272

11.2.1 2-Oxoglutarate Cofactor Mimicking Inhibitors 273

11.2.1.1 Emulation of the Chelating α-Keto AcidMoiety in 2OG 273

11.2.1.2 Bioisosteres of the Conserved 2OG C5-Carboxylic Acid-Binding Motif 273

11.2.2 Histone Substrate-Competitive Inhibitors 275

11.2.2.1 Small-Molecule Inhibitors 276

11.2.2.2 Peptide Inhibitors 276

11.2.3 Allosteric Inhibitors 276

11.2.4 Inhibitors Targeting KDM Subfamilies 277

11.2.4.1 KDM4 Subfamily-Targeted Inhibitors 277

11.2.4.2 KDM4/5 Subfamily-Targeted Inhibitors 279

11.2.4.3 KDM5 Subfamily-Targeted Inhibitors 280

11.2.4.4 KDM6 Subfamily-Targeted Inhibitors 281

11.2.4.5 KDM2/7- and KDM3-Targeted Inhibitors 282

11.2.4.6 Generic JmjC-KDM Inhibitors 282

11.2.5 Selectivity and Potency of JmjC-KDM Inhibition in Cells 283

11.3 KDM Inhibitors Targeting the Reader Domains 284

11.3.1 Plant Homeodomain Fingers (PHD Fingers) 284

11.3.2 Tudor Domains 286

11.4 Conclusions and Future Perspectives 286

Acknowledgments 287

References 287

12 Histone Acetyltransferases: Targets and Inhibitors 297
Gianluca Sbardella

12.1 Introduction 297

12.2 Acetyltransferase Enzymes and Families 298

12.3 The GNAT Superfamily 299

12.3.1 KAT2A/GCN5 and KAT2B/PCAF 301

12.3.2 KAT1/Hat1 303

12.3.3 GCN5L1 304

12.4 KAT3A/CBP and KAT3B/p300 Family 304

12.5 MYST Family 306

12.5.1 KAT5/Tip60 306

12.5.2 KAT6A/MOZ, KAT6B/MORF, and KAT7/HBO1 307

12.5.3 KAT8/MOF 307

12.5.4 SAS2 and SAS3 308

12.5.5 ESA1 308

12.5.6 Other KATs 308

12.6 KATs in Diseases 309

12.7 KAT Modulators 312

12.7.1 Bisubstrate Inhibitors 313

12.7.2 Natural Products and Synthetic Analogues and Derivatives 315

12.7.3 Synthetic Compounds 321

12.7.4 Compounds Targeting Protein–Protein Interaction Domains 328

12.8 Conclusion 333

References 334

13 Bromodomains: Promising Targets for Drug Discovery 347
Mehrosh Pervaiz, PankajMishra, and Stefan Günther

13.1 Introduction 347

13.2 The Human Bromodomain Family 348

13.2.1 Structural Features of the Human BRD Family 348

13.2.1.1 The Kac Binding Site 348

13.2.1.2 Druggability of the Human BRD Family 350

13.2.2 Functions of Bromodomain-containing Proteins 352

13.3 Bromodomains and Diseases 353

13.3.1 The BET Family 354

13.3.2 Non-BET Proteins 356

13.4 Methods for the Identification of Bromodomain Inhibitors 357

13.4.1 High-throughput Screening (HTS) 357

13.4.2 Fragment-based Lead Discovery 359

13.4.3 Structure-based Drug Design 359

13.4.4 Virtual Screening 362

13.4.4.1 Structure-based Virtual Screening 362

13.4.4.2 Ligand-based Virtual Screening 362

13.4.4.3 Pharmacophore Modeling 363

13.4.4.4 Substructure and Similarity Search 363

13.5 Current Bromodomain Inhibitors 364

13.6 Multi-target Inhibitors 365

13.6.1 Dual Kinase–Bromodomain Inhibitors 365

13.6.2 Dual BET/HDAC Inhibitors 369

13.7 Proteolysis Targeting Chimeras (PROTACs) 369

13.8 Conclusions 371

Acknowledgments 372

References 372

14 Lysine Reader Proteins 383
Johannes Bacher, Dina Robaa, Chiara Luise,Wolfgang Sippl, and Manfred Jung

14.1 Introduction 383

14.2 The Royal Family of Epigenetic Reader Proteins 385

14.2.1 The MBT Domain 385

14.2.2 The PWWP Domain 390

14.2.3 The Tudor Domain 392

14.2.4 The Chromodomain 395

14.3 The PHD Finger Family of Epigenetic Reader Proteins 400

14.4 TheWD40 Repeat Domain Family 402

14.5 Conclusion and Outlook 409

Acknowledgment 409

References 409

15 DNA-modifying Enzymes 421
Martin Roatsch, Dina Robaa,Michael Lübbert,Wolfgang Sippl, and Manfred Jung

15.1 Introduction 421

15.2 DNA Methylation 422

15.3 Further Modifications of Cytosine Bases 424

15.4 DNA Methyltransferases: Substrates and Structural Aspects 426

15.5 Mechanism of Enzymatic DNA Methylation 430

15.6 Physiological Role of DNA Methylation 431

15.7 DNA Methylation in Disease 432

15.8 DNMT Inhibitors 433

15.8.1 Nucleoside-mimicking DNMT Inhibitors 433

15.8.2 Non-nucleosidic DNMT Inhibitors 436

15.9 Therapeutic Applications of DNMT Inhibitors 441

15.10 Conclusion 442

Acknowledgment 443

References 443

16 Parasite Epigenetic Targets 457
Raymond J. Pierce and Jamal Khalife

16.1 Introduction: The Global Problem of Parasitic Diseases and the Need for New Drugs 457

16.2 Parasite Epigenetic Mechanisms 458

16.2.1 DNA Methylation 459

16.2.2 Histone Posttranslational Modifications 460

16.2.3 Histone-modifying Enzymes in Parasites 462

16.2.4 HMEs Validated as Therapeutic Targets 462

16.2.5 Structure-based Approaches for Defining Therapeutic Targets 464

16.3 Development of Epi-drugs for Parasitic Diseases 465

16.3.1 Repurposing of Existing Epi-drugs 466

16.3.2 Candidates from Phenotypic or High-throughput Screens 467

16.3.3 Structure-based Development of Selective Inhibitors 467

16.4 Conclusions 468

Acknowledgments 469

References 469

Index 477