Discover a new paradigm in drug discovery that greatly expands the space of addressable drug targets and potential novel drugs
Existing paradigms for drug discovery have focused largely on enzymes and other proteins as drug targets. In recent years, however, different varieties of ribonucleic acids have emerged as a viable focus for target-based drug discovery, with the potential to revolutionize the strategy and approach for this essential step in the drug development process.
RNA as a Drug Target: The Next Frontier for Medicinal Chemistry offers a practice-oriented introduction to developing drug-like small molecules that selectively modulate both coding and non-coding RNAs. Beginning with a description and characterization of existing druggable RNAs, the book discusses how to approach different RNA targets for drug discovery. The result is a crucial resource for targeting RNAs and creating the next generation of life-saving pharmaceuticals.
RNA as a Drug Target readers will also find:
- A complete “toolbox” for working with RNA, from structure determination to screening and lead generation techniques
- A wide range of addressable targets and mechanisms, including splicing modulation, riboswitches, targeted degradation, and more
- Authoritative discussion of the potential of RNA-targeted small molecule therapeutics for drugging the epitranscriptome
RNA as a Drug Target provides an expert introduction to a new frontier in pharmaceutical research for medicinal chemists, biochemists, molecular biologists, and members of the pharmaceutical industry.
Table of Contents
Series Editors’ Preface xiii
Preface xv
1 Introduction 1
John Schneekloth Jr. and Martin Pettersson
References 4
2 RNA Structure Probing, Dynamics, and Folding 7
Danny Incarnato
2.1 Introduction 7
2.1.1 Relevance of RNA Structure in Disease 8
2.1.2 Challenges in Studying RNA Structures 8
2.2 Experimentally Guided RNA Structure Modeling 9
2.2.1 Structural Interrogation of RNA Nucleotides via Chemical Probing 10
2.2.1.1 Limits of RNA Chemical Probing 12
2.2.2 Direct Mapping of RNA-RNA Interactions 14
2.2.2.1 Limits of RNA-RNA Interaction Mapping 16
2.2.3 Mapping Spatially Proximal Nucleotides in RNA molecules 17
2.2.3.1 Limits of Methods for Spatial Proximity Mapping 17
2.3 Dealing with RNA Structure Heterogeneity 19
2.4 Querying RNA-Small Molecule Interactions with Chemical Probing 22
2.5 Conclusions and Future Prospects 22
References 23
3 High-Resolution Structures of RNA 29
Lukas Braun, Zahra Alirezaeizanjani, Roberta Tesch, and Hamed Kooshapur
3.1 Introduction 29
3.2 X-Ray Crystallography 31
3.3 NMR Spectroscopy 34
3.4 Cryo-EM 37
3.5 3D Structure Prediction and Integrative Approaches 39
3.6 Conclusions 43
Acknowledgments 43
Conflicts of Interest 43
References 43
4 Screening and Lead Generation Techniques for RNA Binders 49
Gary Frey, Emily Garcia Sega, and Neil Lajkiewicz
4.1 Knowledge-Based Versus Agnostic Screening 49
4.2 Virtual Screening 50
4.3 Screening Methods 51
4.3.1 High-Throughput Screening (HTS) 51
4.3.1.1 Mass Spectrometry 51
4.3.1.2 HTS of RNA Using Direct MS Approaches 52
4.3.1.3 HTS of RNA Using Indirect MS Approaches 54
4.3.1.4 DNA-Encoded Libraries (DELs) 56
4.3.1.5 Microarray Screening 57
4.3.1.6 Fragment-Based Drug Discovery 58
4.3.1.7 Phage Display 63
4.3.2 Orthogonal Methods 63
4.3.2.1 Surface Plasmon Resonance 63
4.3.2.2 Fluorescence-Based Assays 66
4.3.2.3 Microscale Thermophoresis (MST) 70
4.3.2.4 Isothermal Titration Calorimetry (ITC) 70
4.4 Binding Site Identification/Target Engagement 72
4.4.1 Covalent Methods 72
4.4.2 Competition with an Antisense Oligonucleotide (ASO) 74
4.5 Defining SAR and Functional Assays 75
4.5.1 Functional Assays 75
4.5.2 Phenotypic Screens 76
4.6 Identifying a Lead Series 76
4.6.1 Hit Optimization 77
4.6.2 Risdiplam Hit-to-Lead 78
4.6.3 Branaplam Lead Generation 79
4.6.4 Zotatifin Lead Generation 80
4.7 Concluding Thoughts and Outlook 80
Acknowledgments 81
References 81
5 Chemical Matter That Binds RNA 93
Emily G. Swanson Hay, Zhengguo Cai, and Amanda E. Hargrove
5.1 Introduction 93
5.2 Natural Ligands 94
5.2.1 Aminoglycosides 94
5.2.2 Tetracyclines 95
5.2.3 Macrolides 96
5.2.4 Native Riboswitch Ligands 96
5.3 Commercial Ligands 97
5.3.1 Industrial Libraries 98
5.3.2 Academic Libraries 98
5.4 Synthetic Ligands 99
5.4.1 Benzimidazoles and Purines 100
5.4.2 Naphthalenes, Quinolines, and Quinazolines 101
5.4.3 Oxazolidinones 102
5.4.4 Amilorides 102
5.4.5 Diphenyl Furan 103
5.4.6 Multivalent Ligands 103
5.5 Computational Tools for the Exploration of Chemical Space 103
5.5.1 Similarity Searches and Principal Component Analysis 104
5.5.2 Additional Machine-Learning Tools 105
5.5.3 Structure-Based Ligand Design 106
5.6 Case Studies in Examining and Expanding RNA-Targeted Chemical Space 106
5.6.1 Using QSAR to Probe RNA-Targeting Small-Molecule Properties 107
5.6.2 Evaluating the Chemical Space of Natural, Synthetic, and Commercial Ligands 108
5.7 Conclusions and Outlook 111
Acknowledgments 111
References 111
6 MicroRNAs as Targets for Small-Molecule Binders 119
Maria Duca
6.1 Introduction 119
6.2 MicroRNAs 121
6.3 MicroRNAs Biogenesis 122
6.4 Targeting MicroRNAs with Small-Molecule RNA Binders 123
6.4.1 Induction of miRNAs Expression: Tackling the Decrease of Tumor Suppressor miRNAs 124
6.4.2 Inhibition of miRNAs Production: Pre- and Pri-miRNA Binders 125
6.4.2.1 Discovery of miRNAs Inhibitors by Intracellular Assays 125
6.4.2.2 Target-Based In Vitro Assays 127
6.4.2.3 Design of Specific Ligands of Pre- and Pri-miRNAs 131
6.4.2.4 Fragment-Based Drug Design 138
6.4.2.5 DNA-Encoded Libraries (DELs) 139
6.5 Inhibition of RNA-Protein Interactions in miRNAs Pathways 140
6.6 Adding Cleavage Properties to miRNAs Interfering Agents 142
6.7 Conclusions 144
References 144
7 Pre-mRNA Splicing Modulation 151
Scott J. Barraza and Matthew G. Woll
7.1 Introduction 151
7.2 Overview of Splicing Biology 152
7.2.1 The Spliceosome 152
7.2.2 Classes of Alternative Splicing 154
7.3 Pharmacological Mechanisms of Splicing Modulation 155
7.3.1 Cis- and Trans-Regulatory Elements (Splicing Factors) 155
7.3.1.1 Stabilization of Cis-Regulatory Elements 156
7.3.1.2 Destabilization of Cis-Regulatory Elements 158
7.3.1.3 Inhibition of Cis-Regulatory RNA-Protein Interactions 158
7.3.1.4 Inhibition of Trans-Regulatory Elements 160
7.3.1.5 Degradation of Trans-Regulatory Elements 161
7.3.1.6 Inhibition of Trans-Regulatory Element Protein-Protein Interactions (PPIs) 162
7.3.1.7 Stabilization of Trans-Regulatory Element RNA-Protein Interactions (RPIs) 165
7.3.2 Kinases and Phosphatases 165
7.3.2.1 Challenges in Targeting Kinases 167
7.3.2.2 Inhibition of Kinases 168
7.3.2.3 Activation and Degradation of Kinases 168
7.3.2.4 Inhibition and Activation of Protein Phosphatases 169
7.3.3 Epigenetic Writers and Erasers 172
7.3.3.1 Inhibition of Epigenetic Writers 172
7.3.4 RNA Helicases 174
7.3.5 Drugging the Spliceosome 175
7.3.5.1 Inhibition of U2 snRNP Recognition of the 3′-Splice Site 176
7.3.5.2 E7107 176
7.3.5.3 H3B-8800 177
7.3.5.4 Stabilizers of U1 snRNP Recognition of the 5′-Splice Site 180
7.3.5.5 Introduction to Spinal Muscular Atrophy (SMA) 180
7.3.5.6 Risdiplam (Evrysdi®) 183
7.4 Future Outlook 186
References 188
8 Prospects for Riboswitches in Drug Development 203
Michael G. Mohsen and Ronald R. Breaker
8.1 Introduction 203
8.1.1 The Known Landscape of Riboswitches 203
8.1.2 Riboswitches in Drug Development 203
8.1.3 The Need for Novel Antibiotics 205
8.2 Riboswitches as Drug Targets 207
8.2.1 Why Target Riboswitches? 207
8.2.2 Features of a Druggable Riboswitch 208
8.2.3 Riboswitch-Targeted Drugs 208
8.2.3.1 Small Molecules Targeting FMN Riboswitches 208
8.2.3.2 Other Riboswitches Targeted in Proof-of-Principle Demonstrations 209
8.2.4 Barriers and Future Developments 210
8.3 Riboswitches as Tools for Antibiotic Drug Development 210
8.3.1 Riboswitches as Biosensors 210
8.3.2 A Riboswitch-Based Fluoride Sensor Illuminates Agonists of Fluoride Toxicity 211
8.3.3 A Riboswitch-Based ZTP Sensor Identifies Inhibitors of Folate Biosynthesis 211
8.3.4 A Riboswitch-Based SAH Sensor Reveals an Inhibitor of SAH Nucleosidase 212
8.3.5 Barriers and Future Developments 213
8.4 Application of Riboswitches in Gene Therapy 213
8.4.1 Considerations for Designer Riboswitches 213
8.4.2 Eukaryotic Expression Platforms 214
8.4.3 Barriers and Future Developments 216
8.5 Concluding Remarks 217
Acknowledgment 218
References 218
9 Small Molecules That Degrade RNA 227
Noah A. Springer, Samantha M. Meyer, Amirhossein Taghavi, Jessica L. Childs-Disney, and Matthew D. Disney
9.1 Antisense Oligonucleotide Degraders 227
9.2 Small-Molecule Direct Degraders 228
9.2.1 N-Hydroxypyridine-2(1H)-thione (N-HPT) Conjugates 229
9.2.2 Bleomycin 229
9.2.3 Bleomycin Conjugates 231
9.2.3.1 Bleomycin Degraders Targeting the r(CUG) Repeat Expansion That Causes DM1 231
9.2.3.2 Bleomycin Degraders Targeting r(CCUG) Repeat Expansion that Causes DM2 233
9.2.3.3 Bleomycin Degraders Targeting Oncogenic Precursor microRNAs 233
9.2.3.4 Conclusions and Outlook for Bleomycin-Based Direct Degraders 234
9.3 Ribonuclease Targeting Chimeras (RiboTACs) 235
9.3.1 RNase L is an Endogenous Endoribonuclease That Functions as Part of the Innate Immune Response 236
9.3.2 First-Generation RiboTACs Targeting Oncogenic miRNAs 236
9.3.3 Small-Molecule-Based RiboTACs 239
9.3.4 Comparison of Bleomycin-Based Direct Degraders and RiboTACs 242
9.3.5 Discovery of Additional Small-Molecule RNase L Activators 242
9.3.6 Conclusions and Outlook for RiboTACs 243
9.4 Summary and Outlook for Small-Molecule RNA Degraders 244
References 246
10 Approaches to the Identification of Molecules Altering Programmed Ribosomal Frameshifting in Viruses 253
Elinore A. VanGraafeiland, Diego M. Arévalo, and Benjamin L. Miller
10.1 Introduction 253
10.2 Mechanisms of Frameshifting 256
10.3 Targeting Frameshifting in HIV 257
10.4 Targeting Frameshifting in SARS-CoV-1 and SARS-CoV-2 263
10.5 Conclusions 274
References 274
11 RNA-Protein Interactions: A New Approach for Drugging RNA Biology 281
Dalia M. Soueid and Amanda L. Garner
11.1 Molecular Basis of RNA-Protein Interactions 282
11.1.1 RNA Recognition Motifs (RRMs) 282
11.1.2 Double-Stranded RNA-Binding Domains (dsRBD) 286
11.1.3 Zinc Finger (ZnF) Domains 287
11.1.4 K Homology (KH) Domains 289
11.1.5 Other RBDs 290
11.2 Regulation and Dysregulation of RNA-Protein Interactions 290
11.2.1 Poor Quality Control Leads to Over- and Underproduction of RBPs 292
11.2.2 RBPs Become Out of Control, mRNA Processing Gets a Makeover (and Hates It) 294
11.2.3 RBP Shuttling of mRNA Becomes Askew 294
11.2.4 The RBP is Lost and Wreaks Havoc on the Cell 295
11.2.5 RBPs Dictate Which mRNAs are Translated, Favoring their Toxic Friends 295
11.2.6 RBPs and RNA Become Very Clique-y, Form Their Own Complex and Cause Stress to the Rest of the Cell 296
11.3 Experimental Methods to Detect and Screen for Small Molecules that Modulate RNA-Protein Interactions 297
11.3.1 In vitro Fluorescence-Based Assays 297
11.3.2 In vitro Chemiluminescence-Based Assays 297
11.3.2.1 Cell-Based RPI Detection Assays 300
11.3.3 Cell-Based RNA-Protein Interaction Screening 301
11.4 Closing Remarks 302
References 303
12 Drugging the Epitranscriptome 321
Tanner W. Eggert and Ralph E. Kleiner
12.1 Introduction 321
12.2 Modifications on mRNA: N6-Methyladenosine, Pseudouridine, and Inosine 325
12.2.1 N6-Methyladenosine (m 6 A) 325
12.2.2 Pseudouridine (Ψ) 327
12.2.3 Inosine (I) 328
12.3 Modifications on tRNA and rRNA 330
12.3.1 tRNA Modifications 330
12.3.2 rRNA Modifications 334
12.4 Concluding Remarks 335
References 336
13 Outlook 355
Christopher R. Fullenkamp, Xiao Liang, Martin Pettersson, and John Schneekloth Jr.
13.1 Introduction 355
13.2 Target Selection: Identification of the Most Promising RNA Intervention Points 357
13.3 Development of Robust Biophysical Methods, Alternative Strategies for Target Engagement, and Accurate and Reliable Functional Models 358
13.3.1 Biophysical Methods for Interrogating Small Molecule-RNA Interactions 358
13.3.2 Cellular Target Engagement Methods 360
13.3.3 Unique Challenges Faced in the Development of Functional Assays for Studying Small Molecule-RNA Interactions 364
13.4 Acquisition of High-Resolution RNA and RNA-Ligand Structures is Needed to Enable the Development and Validation of Computational Tools for RNA-Small Molecule Therapeutic Discovery 367
13.4.1 RNA Structure Prediction 367
13.4.2 Computational Tools for Hit Optimization 369
13.4.3 Implementation of Molecular Dynamics Simulations, Machine Learning, and AI Tools to Interrogate RNA-Small Molecule Interactions 371
13.5 Deposition of Small Molecule-RNA Interaction Data with Rigorous Experimental Protocols and Controls is Needed 373
13.6 Outlook: The Future of Small Molecule-Based RNA Therapeutics is Bright 375
References 376
Index 385
