John Schneekloth, Jr., PhD (Jay), obtained his PhD from the chemistry department of Yale University. Following postdoctoral work at Princeton, Dr. Schneekloth joined the NCI, where his research involves using synthetic chemistry and high throughput chemical biology approaches to develop chemical probes of RNA, with a particular emphasis on targeting RNA with druglike small molecules.Martin Pettersson, PhD, obtained his PhD in Organic Chemistry from the University of Texas at Austin. He is a medical chemistry / drug discovery leader with over 20 years of industrial experience, mainly with Pfizer (co-inventor of Paxlovid). He has then continued as Senior Director of Neuroscience/Pain, Chemistry, at Grünenthal Boston Innovation Hub, and most recently as VP of Drug Discovery at Promedigen in South Korea.

• Series Editors’ Preface xiiiPreface xv1 Introduction 1John Schneekloth Jr. and Martin PetterssonReferences 42 RNA Structure Probing, Dynamics, and Folding 7Danny Incarnato2.1 Introduction 72.1.1 Relevance of RNA Structure in Disease 82.1.2 Challenges in Studying RNA Structures 82.2 Experimentally Guided RNA Structure Modeling 92.2.1 Structural Interrogation of RNA Nucleotides via Chemical Probing 102.2.1.1 Limits of RNA Chemical Probing 122.2.2 Direct Mapping of RNA–RNA Interactions 142.2.2.1 Limits of RNA–RNA Interaction Mapping 162.2.3 Mapping Spatially Proximal Nucleotides in RNA molecules 172.2.3.1 Limits of Methods for Spatial Proximity Mapping 172.3 Dealing with RNA Structure Heterogeneity 192.4 Querying RNA–Small Molecule Interactions with Chemical Probing 222.5 Conclusions and Future Prospects 22References 233 High-Resolution Structures of RNA 29Lukas Braun, Zahra Alirezaeizanjani, Roberta Tesch, and Hamed Kooshapur3.1 Introduction 293.2 X-Ray Crystallography 313.3 NMR Spectroscopy 343.4 Cryo-EM 373.5 3D Structure Prediction and Integrative Approaches 393.6 Conclusions 43Acknowledgments 43Conflicts of Interest 43References 434 Screening and Lead Generation Techniques for RNA Binders 49Gary Frey, Emily Garcia Sega, and Neil Lajkiewicz4.1 Knowledge-Based Versus Agnostic Screening 494.2 Virtual Screening 504.3 Screening Methods 514.3.1 High-Throughput Screening (HTS) 514.3.1.1 Mass Spectrometry 514.3.1.2 HTS of RNA Using Direct MS Approaches 524.3.1.3 HTS of RNA Using Indirect MS Approaches 544.3.1.4 DNA-Encoded Libraries (DELs) 564.3.1.5 Microarray Screening 574.3.1.6 Fragment-Based Drug Discovery 584.3.1.7 Phage Display 634.3.2 Orthogonal Methods 634.3.2.1 Surface Plasmon Resonance 634.3.2.2 Fluorescence-Based Assays 664.3.2.3 Microscale Thermophoresis (MST) 704.3.2.4 Isothermal Titration Calorimetry (ITC) 704.4 Binding Site Identification/Target Engagement 724.4.1 Covalent Methods 724.4.2 Competition with an Antisense Oligonucleotide (ASO) 744.5 Defining SAR and Functional Assays 754.5.1 Functional Assays 754.5.2 Phenotypic Screens 764.6 Identifying a Lead Series 764.6.1 Hit Optimization 774.6.2 Risdiplam Hit-to-Lead 784.6.3 Branaplam Lead Generation 794.6.4 Zotatifin Lead Generation 804.7 Concluding Thoughts and Outlook 80Acknowledgments 81References 815 Chemical Matter That Binds RNA 93Emily G. Swanson Hay, Zhengguo Cai, and Amanda E. Hargrove5.1 Introduction 935.2 Natural Ligands 945.2.1 Aminoglycosides 945.2.2 Tetracyclines 955.2.3 Macrolides 965.2.4 Native Riboswitch Ligands 965.3 Commercial Ligands 975.3.1 Industrial Libraries 985.3.2 Academic Libraries 985.4 Synthetic Ligands 995.4.1 Benzimidazoles and Purines 1005.4.2 Naphthalenes, Quinolines, and Quinazolines 1015.4.3 Oxazolidinones 1025.4.4 Amilorides 1025.4.5 Diphenyl Furan 1035.4.6 Multivalent Ligands 1035.5 Computational Tools for the Exploration of Chemical Space 1035.5.1 Similarity Searches and Principal Component Analysis 1045.5.2 Additional Machine-Learning Tools 1055.5.3 Structure-Based Ligand Design 1065.6 Case Studies in Examining and Expanding RNA-Targeted Chemical Space 1065.6.1 Using QSAR to Probe RNA-Targeting Small-Molecule Properties 1075.6.2 Evaluating the Chemical Space of Natural, Synthetic, and Commercial Ligands 1085.7 Conclusions and Outlook 111Acknowledgments 111References 1116 MicroRNAs as Targets for Small-Molecule Binders 119Maria Duca6.1 Introduction 1196.2 MicroRNAs 1216.3 MicroRNAs Biogenesis 1226.4 Targeting MicroRNAs with Small-Molecule RNA Binders 1236.4.1 Induction of miRNAs Expression: Tackling the Decrease of Tumor Suppressor miRNAs 1246.4.2 Inhibition of miRNAs Production: Pre- and Pri-miRNA Binders 1256.4.2.1 Discovery of miRNAs Inhibitors by Intracellular Assays 1256.4.2.2 Target-Based In Vitro Assays 1276.4.2.3 Design of Specific Ligands of Pre- and Pri-miRNAs 1316.4.2.4 Fragment-Based Drug Design 1386.4.2.5 DNA-Encoded Libraries (DELs) 1396.5 Inhibition of RNA–Protein Interactions in miRNAs Pathways 1406.6 Adding Cleavage Properties to miRNAs Interfering Agents 1426.7 Conclusions 144References 1447 Pre-mRNA Splicing Modulation 151Scott J. Barraza and Matthew G. Woll7.1 Introduction 1517.2 Overview of Splicing Biology 1527.2.1 The Spliceosome 1527.2.2 Classes of Alternative Splicing 1547.3 Pharmacological Mechanisms of Splicing Modulation 1557.3.1 Cis- and Trans-Regulatory Elements (Splicing Factors) 1557.3.1.1 Stabilization of Cis-Regulatory Elements 1567.3.1.2 Destabilization of Cis-Regulatory Elements 1587.3.1.3 Inhibition of Cis-Regulatory RNA–Protein Interactions 1587.3.1.4 Inhibition of Trans-Regulatory Elements 1607.3.1.5 Degradation of Trans-Regulatory Elements 1617.3.1.6 Inhibition of Trans-Regulatory Element Protein–Protein Interactions (PPIs) 1627.3.1.7 Stabilization of Trans-Regulatory Element RNA–Protein Interactions (RPIs) 1657.3.2 Kinases and Phosphatases 1657.3.2.1 Challenges in Targeting Kinases 1677.3.2.2 Inhibition of Kinases 1687.3.2.3 Activation and Degradation of Kinases 1687.3.2.4 Inhibition and Activation of Protein Phosphatases 1697.3.3 Epigenetic Writers and Erasers 1727.3.3.1 Inhibition of Epigenetic Writers 1727.3.4 RNA Helicases 1747.3.5 Drugging the Spliceosome 1757.3.5.1 Inhibition of U2 snRNP Recognition of the 3′-Splice Site 1767.3.5.2 E7107 1767.3.5.3 H3B-8800 1777.3.5.4 Stabilizers of U1 snRNP Recognition of the 5′-Splice Site 1807.3.5.5 Introduction to Spinal Muscular Atrophy (SMA) 1807.3.5.6 Risdiplam (Evrysdi®) 1837.4 Future Outlook 186References 1888 Prospects for Riboswitches in Drug Development 203Michael G. Mohsen and Ronald R. Breaker8.1 Introduction 2038.1.1 The Known Landscape of Riboswitches 2038.1.2 Riboswitches in Drug Development 2038.1.3 The Need for Novel Antibiotics 2058.2 Riboswitches as Drug Targets 2078.2.1 Why Target Riboswitches? 2078.2.2 Features of a Druggable Riboswitch 2088.2.3 Riboswitch-Targeted Drugs 2088.2.3.1 Small Molecules Targeting FMN Riboswitches 2088.2.3.2 Other Riboswitches Targeted in Proof-of-Principle Demonstrations 2098.2.4 Barriers and Future Developments 2108.3 Riboswitches as Tools for Antibiotic Drug Development 2108.3.1 Riboswitches as Biosensors 2108.3.2 A Riboswitch-Based Fluoride Sensor Illuminates Agonists of Fluoride Toxicity 2118.3.3 A Riboswitch-Based ZTP Sensor Identifies Inhibitors of Folate Biosynthesis 2118.3.4 A Riboswitch-Based SAH Sensor Reveals an Inhibitor of SAH Nucleosidase 2128.3.5 Barriers and Future Developments 2138.4 Application of Riboswitches in Gene Therapy 2138.4.1 Considerations for Designer Riboswitches 2138.4.2 Eukaryotic Expression Platforms 2148.4.3 Barriers and Future Developments 2168.5 Concluding Remarks 217Acknowledgment 218References 2189 Small Molecules That Degrade RNA 227Noah A. Springer, Samantha M. Meyer, Amirhossein Taghavi, Jessica L. Childs-Disney, and Matthew D. Disney9.1 Antisense Oligonucleotide Degraders 2279.2 Small-Molecule Direct Degraders 2289.2.1 N-Hydroxypyridine-2(1H)-thione (N-HPT) Conjugates 2299.2.2 Bleomycin 2299.2.3 Bleomycin Conjugates 2319.2.3.1 Bleomycin Degraders Targeting the r(CUG) Repeat Expansion That Causes DM1 2319.2.3.2 Bleomycin Degraders Targeting r(CCUG) Repeat Expansion that Causes DM2 2339.2.3.3 Bleomycin Degraders Targeting Oncogenic Precursor microRNAs 2339.2.3.4 Conclusions and Outlook for Bleomycin-Based Direct Degraders 2349.3 Ribonuclease Targeting Chimeras (RiboTACs) 2359.3.1 RNase L is an Endogenous Endoribonuclease That Functions as Part of the Innate Immune Response 2369.3.2 First-Generation RiboTACs Targeting Oncogenic miRNAs 2369.3.3 Small-Molecule-Based RiboTACs 2399.3.4 Comparison of Bleomycin-Based Direct Degraders and RiboTACs 2429.3.5 Discovery of Additional Small-Molecule RNase L Activators 2429.3.6 Conclusions and Outlook for RiboTACs 2439.4 Summary and Outlook for Small-Molecule RNA Degraders 244References 24610 Approaches to the Identification of Molecules Altering Programmed Ribosomal Frameshifting in Viruses 253Elinore A. VanGraafeiland, Diego M. Arévalo, and Benjamin L. Miller10.1 Introduction 25310.2 Mechanisms of Frameshifting 25610.3 Targeting Frameshifting in HIV 25710.4 Targeting Frameshifting in SARS-CoV-1 and SARS-CoV-2 26310.5 Conclusions 274References 27411 RNA–Protein Interactions: A New Approach for Drugging RNA Biology 281Dalia M. Soueid and Amanda L. Garner11.1 Molecular Basis of RNA–Protein Interactions 28211.1.1 RNA Recognition Motifs (RRMs) 28211.1.2 Double-Stranded RNA-Binding Domains (dsRBD) 28611.1.3 Zinc Finger (ZnF) Domains 28711.1.4 K Homology (KH) Domains 28911.1.5 Other RBDs 29011.2 Regulation and Dysregulation of RNA–Protein Interactions 29011.2.1 Poor Quality Control Leads to Over- and Underproduction of RBPs 29211.2.2 RBPs Become Out of Control, mRNA Processing Gets a Makeover (and Hates It) 29411.2.3 RBP Shuttling of mRNA Becomes Askew 29411.2.4 The RBP is Lost and Wreaks Havoc on the Cell 29511.2.5 RBPs Dictate Which mRNAs are Translated, Favoring their Toxic Friends 29511.2.6 RBPs and RNA Become Very Clique-y, Form Their Own Complex and Cause Stress to the Rest of the Cell 29611.3 Experimental Methods to Detect and Screen for Small Molecules that Modulate RNA–Protein Interactions 29711.3.1 In vitro Fluorescence-Based Assays 29711.3.2 In vitro Chemiluminescence-Based Assays 29711.3.2.1 Cell-Based RPI Detection Assays 30011.3.3 Cell-Based RNA–Protein Interaction Screening 30111.4 Closing Remarks 302References 30312 Drugging the Epitranscriptome 321Tanner W. Eggert and Ralph E. Kleiner12.1 Introduction 32112.2 Modifications on mRNA: N6-Methyladenosine, Pseudouridine, and Inosine 32512.2.1 N6-Methyladenosine (m 6 A) 32512.2.2 Pseudouridine (Ψ) 32712.2.3 Inosine (I) 32812.3 Modifications on tRNA and rRNA 33012.3.1 tRNA Modifications 33012.3.2 rRNA Modifications 33412.4 Concluding Remarks 335References 33613 Outlook 355Christopher R. Fullenkamp, Xiao Liang, Martin Pettersson, and John Schneekloth Jr.13.1 Introduction 35513.2 Target Selection: Identification of the Most Promising RNA Intervention Points 35713.3 Development of Robust Biophysical Methods, Alternative Strategies for Target Engagement, and Accurate and Reliable Functional Models 35813.3.1 Biophysical Methods for Interrogating Small Molecule–RNA Interactions 35813.3.2 Cellular Target Engagement Methods 36013.3.3 Unique Challenges Faced in the Development of Functional Assays for Studying Small Molecule–RNA Interactions 36413.4 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 36713.4.1 RNA Structure Prediction 36713.4.2 Computational Tools for Hit Optimization 36913.4.3 Implementation of Molecular Dynamics Simulations, Machine Learning, and AI Tools to Interrogate RNA–Small Molecule Interactions 37113.5 Deposition of Small Molecule–RNA Interaction Data with Rigorous Experimental Protocols and Controls is Needed 37313.6 Outlook: The Future of Small Molecule-Based RNA Therapeutics is Bright 375References 376Index 385

John Schneekloth, Jr., PhD (Jay), obtained his PhD from the chemistry department of Yale University. Following postdoctoral work at Princeton, Dr. Schneekloth joined the NCI, where his research involves using synthetic chemistry and high throughput chemical biology approaches to develop chemical probes of RNA, with a particular emphasis on targeting RNA with druglike small molecules.Martin Pettersson, PhD, obtained his PhD in Organic Chemistry from the University of Texas at Austin. He is a medical chemistry / drug discovery leader with over 20 years of industrial experience, mainly with Pfizer (co-inventor of Paxlovid). He has then continued as Senior Director of Neuroscience/Pain, Chemistry, at Grünenthal Boston Innovation Hub, and most recently as VP of Drug Discovery at Promedigen in South Korea.

• Series Editors’ Preface xiiiPreface xv1 Introduction 1John Schneekloth Jr. and Martin PetterssonReferences 42 RNA Structure Probing, Dynamics, and Folding 7Danny Incarnato2.1 Introduction 72.1.1 Relevance of RNA Structure in Disease 82.1.2 Challenges in Studying RNA Structures 82.2 Experimentally Guided RNA Structure Modeling 92.2.1 Structural Interrogation of RNA Nucleotides via Chemical Probing 102.2.1.1 Limits of RNA Chemical Probing 122.2.2 Direct Mapping of RNA–RNA Interactions 142.2.2.1 Limits of RNA–RNA Interaction Mapping 162.2.3 Mapping Spatially Proximal Nucleotides in RNA molecules 172.2.3.1 Limits of Methods for Spatial Proximity Mapping 172.3 Dealing with RNA Structure Heterogeneity 192.4 Querying RNA–Small Molecule Interactions with Chemical Probing 222.5 Conclusions and Future Prospects 22References 233 High-Resolution Structures of RNA 29Lukas Braun, Zahra Alirezaeizanjani, Roberta Tesch, and Hamed Kooshapur3.1 Introduction 293.2 X-Ray Crystallography 313.3 NMR Spectroscopy 343.4 Cryo-EM 373.5 3D Structure Prediction and Integrative Approaches 393.6 Conclusions 43Acknowledgments 43Conflicts of Interest 43References 434 Screening and Lead Generation Techniques for RNA Binders 49Gary Frey, Emily Garcia Sega, and Neil Lajkiewicz4.1 Knowledge-Based Versus Agnostic Screening 494.2 Virtual Screening 504.3 Screening Methods 514.3.1 High-Throughput Screening (HTS) 514.3.1.1 Mass Spectrometry 514.3.1.2 HTS of RNA Using Direct MS Approaches 524.3.1.3 HTS of RNA Using Indirect MS Approaches 544.3.1.4 DNA-Encoded Libraries (DELs) 564.3.1.5 Microarray Screening 574.3.1.6 Fragment-Based Drug Discovery 584.3.1.7 Phage Display 634.3.2 Orthogonal Methods 634.3.2.1 Surface Plasmon Resonance 634.3.2.2 Fluorescence-Based Assays 664.3.2.3 Microscale Thermophoresis (MST) 704.3.2.4 Isothermal Titration Calorimetry (ITC) 704.4 Binding Site Identification/Target Engagement 724.4.1 Covalent Methods 724.4.2 Competition with an Antisense Oligonucleotide (ASO) 744.5 Defining SAR and Functional Assays 754.5.1 Functional Assays 754.5.2 Phenotypic Screens 764.6 Identifying a Lead Series 764.6.1 Hit Optimization 774.6.2 Risdiplam Hit-to-Lead 784.6.3 Branaplam Lead Generation 794.6.4 Zotatifin Lead Generation 804.7 Concluding Thoughts and Outlook 80Acknowledgments 81References 815 Chemical Matter That Binds RNA 93Emily G. Swanson Hay, Zhengguo Cai, and Amanda E. Hargrove5.1 Introduction 935.2 Natural Ligands 945.2.1 Aminoglycosides 945.2.2 Tetracyclines 955.2.3 Macrolides 965.2.4 Native Riboswitch Ligands 965.3 Commercial Ligands 975.3.1 Industrial Libraries 985.3.2 Academic Libraries 985.4 Synthetic Ligands 995.4.1 Benzimidazoles and Purines 1005.4.2 Naphthalenes, Quinolines, and Quinazolines 1015.4.3 Oxazolidinones 1025.4.4 Amilorides 1025.4.5 Diphenyl Furan 1035.4.6 Multivalent Ligands 1035.5 Computational Tools for the Exploration of Chemical Space 1035.5.1 Similarity Searches and Principal Component Analysis 1045.5.2 Additional Machine-Learning Tools 1055.5.3 Structure-Based Ligand Design 1065.6 Case Studies in Examining and Expanding RNA-Targeted Chemical Space 1065.6.1 Using QSAR to Probe RNA-Targeting Small-Molecule Properties 1075.6.2 Evaluating the Chemical Space of Natural, Synthetic, and Commercial Ligands 1085.7 Conclusions and Outlook 111Acknowledgments 111References 1116 MicroRNAs as Targets for Small-Molecule Binders 119Maria Duca6.1 Introduction 1196.2 MicroRNAs 1216.3 MicroRNAs Biogenesis 1226.4 Targeting MicroRNAs with Small-Molecule RNA Binders 1236.4.1 Induction of miRNAs Expression: Tackling the Decrease of Tumor Suppressor miRNAs 1246.4.2 Inhibition of miRNAs Production: Pre- and Pri-miRNA Binders 1256.4.2.1 Discovery of miRNAs Inhibitors by Intracellular Assays 1256.4.2.2 Target-Based In Vitro Assays 1276.4.2.3 Design of Specific Ligands of Pre- and Pri-miRNAs 1316.4.2.4 Fragment-Based Drug Design 1386.4.2.5 DNA-Encoded Libraries (DELs) 1396.5 Inhibition of RNA–Protein Interactions in miRNAs Pathways 1406.6 Adding Cleavage Properties to miRNAs Interfering Agents 1426.7 Conclusions 144References 1447 Pre-mRNA Splicing Modulation 151Scott J. Barraza and Matthew G. Woll7.1 Introduction 1517.2 Overview of Splicing Biology 1527.2.1 The Spliceosome 1527.2.2 Classes of Alternative Splicing 1547.3 Pharmacological Mechanisms of Splicing Modulation 1557.3.1 Cis- and Trans-Regulatory Elements (Splicing Factors) 1557.3.1.1 Stabilization of Cis-Regulatory Elements 1567.3.1.2 Destabilization of Cis-Regulatory Elements 1587.3.1.3 Inhibition of Cis-Regulatory RNA–Protein Interactions 1587.3.1.4 Inhibition of Trans-Regulatory Elements 1607.3.1.5 Degradation of Trans-Regulatory Elements 1617.3.1.6 Inhibition of Trans-Regulatory Element Protein–Protein Interactions (PPIs) 1627.3.1.7 Stabilization of Trans-Regulatory Element RNA–Protein Interactions (RPIs) 1657.3.2 Kinases and Phosphatases 1657.3.2.1 Challenges in Targeting Kinases 1677.3.2.2 Inhibition of Kinases 1687.3.2.3 Activation and Degradation of Kinases 1687.3.2.4 Inhibition and Activation of Protein Phosphatases 1697.3.3 Epigenetic Writers and Erasers 1727.3.3.1 Inhibition of Epigenetic Writers 1727.3.4 RNA Helicases 1747.3.5 Drugging the Spliceosome 1757.3.5.1 Inhibition of U2 snRNP Recognition of the 3′-Splice Site 1767.3.5.2 E7107 1767.3.5.3 H3B-8800 1777.3.5.4 Stabilizers of U1 snRNP Recognition of the 5′-Splice Site 1807.3.5.5 Introduction to Spinal Muscular Atrophy (SMA) 1807.3.5.6 Risdiplam (Evrysdi®) 1837.4 Future Outlook 186References 1888 Prospects for Riboswitches in Drug Development 203Michael G. Mohsen and Ronald R. Breaker8.1 Introduction 2038.1.1 The Known Landscape of Riboswitches 2038.1.2 Riboswitches in Drug Development 2038.1.3 The Need for Novel Antibiotics 2058.2 Riboswitches as Drug Targets 2078.2.1 Why Target Riboswitches? 2078.2.2 Features of a Druggable Riboswitch 2088.2.3 Riboswitch-Targeted Drugs 2088.2.3.1 Small Molecules Targeting FMN Riboswitches 2088.2.3.2 Other Riboswitches Targeted in Proof-of-Principle Demonstrations 2098.2.4 Barriers and Future Developments 2108.3 Riboswitches as Tools for Antibiotic Drug Development 2108.3.1 Riboswitches as Biosensors 2108.3.2 A Riboswitch-Based Fluoride Sensor Illuminates Agonists of Fluoride Toxicity 2118.3.3 A Riboswitch-Based ZTP Sensor Identifies Inhibitors of Folate Biosynthesis 2118.3.4 A Riboswitch-Based SAH Sensor Reveals an Inhibitor of SAH Nucleosidase 2128.3.5 Barriers and Future Developments 2138.4 Application of Riboswitches in Gene Therapy 2138.4.1 Considerations for Designer Riboswitches 2138.4.2 Eukaryotic Expression Platforms 2148.4.3 Barriers and Future Developments 2168.5 Concluding Remarks 217Acknowledgment 218References 2189 Small Molecules That Degrade RNA 227Noah A. Springer, Samantha M. Meyer, Amirhossein Taghavi, Jessica L. Childs-Disney, and Matthew D. Disney9.1 Antisense Oligonucleotide Degraders 2279.2 Small-Molecule Direct Degraders 2289.2.1 N-Hydroxypyridine-2(1H)-thione (N-HPT) Conjugates 2299.2.2 Bleomycin 2299.2.3 Bleomycin Conjugates 2319.2.3.1 Bleomycin Degraders Targeting the r(CUG) Repeat Expansion That Causes DM1 2319.2.3.2 Bleomycin Degraders Targeting r(CCUG) Repeat Expansion that Causes DM2 2339.2.3.3 Bleomycin Degraders Targeting Oncogenic Precursor microRNAs 2339.2.3.4 Conclusions and Outlook for Bleomycin-Based Direct Degraders 2349.3 Ribonuclease Targeting Chimeras (RiboTACs) 2359.3.1 RNase L is an Endogenous Endoribonuclease That Functions as Part of the Innate Immune Response 2369.3.2 First-Generation RiboTACs Targeting Oncogenic miRNAs 2369.3.3 Small-Molecule-Based RiboTACs 2399.3.4 Comparison of Bleomycin-Based Direct Degraders and RiboTACs 2429.3.5 Discovery of Additional Small-Molecule RNase L Activators 2429.3.6 Conclusions and Outlook for RiboTACs 2439.4 Summary and Outlook for Small-Molecule RNA Degraders 244References 24610 Approaches to the Identification of Molecules Altering Programmed Ribosomal Frameshifting in Viruses 253Elinore A. VanGraafeiland, Diego M. Arévalo, and Benjamin L. Miller10.1 Introduction 25310.2 Mechanisms of Frameshifting 25610.3 Targeting Frameshifting in HIV 25710.4 Targeting Frameshifting in SARS-CoV-1 and SARS-CoV-2 26310.5 Conclusions 274References 27411 RNA–Protein Interactions: A New Approach for Drugging RNA Biology 281Dalia M. Soueid and Amanda L. Garner11.1 Molecular Basis of RNA–Protein Interactions 28211.1.1 RNA Recognition Motifs (RRMs) 28211.1.2 Double-Stranded RNA-Binding Domains (dsRBD) 28611.1.3 Zinc Finger (ZnF) Domains 28711.1.4 K Homology (KH) Domains 28911.1.5 Other RBDs 29011.2 Regulation and Dysregulation of RNA–Protein Interactions 29011.2.1 Poor Quality Control Leads to Over- and Underproduction of RBPs 29211.2.2 RBPs Become Out of Control, mRNA Processing Gets a Makeover (and Hates It) 29411.2.3 RBP Shuttling of mRNA Becomes Askew 29411.2.4 The RBP is Lost and Wreaks Havoc on the Cell 29511.2.5 RBPs Dictate Which mRNAs are Translated, Favoring their Toxic Friends 29511.2.6 RBPs and RNA Become Very Clique-y, Form Their Own Complex and Cause Stress to the Rest of the Cell 29611.3 Experimental Methods to Detect and Screen for Small Molecules that Modulate RNA–Protein Interactions 29711.3.1 In vitro Fluorescence-Based Assays 29711.3.2 In vitro Chemiluminescence-Based Assays 29711.3.2.1 Cell-Based RPI Detection Assays 30011.3.3 Cell-Based RNA–Protein Interaction Screening 30111.4 Closing Remarks 302References 30312 Drugging the Epitranscriptome 321Tanner W. Eggert and Ralph E. Kleiner12.1 Introduction 32112.2 Modifications on mRNA: N6-Methyladenosine, Pseudouridine, and Inosine 32512.2.1 N6-Methyladenosine (m 6 A) 32512.2.2 Pseudouridine (Ψ) 32712.2.3 Inosine (I) 32812.3 Modifications on tRNA and rRNA 33012.3.1 tRNA Modifications 33012.3.2 rRNA Modifications 33412.4 Concluding Remarks 335References 33613 Outlook 355Christopher R. Fullenkamp, Xiao Liang, Martin Pettersson, and John Schneekloth Jr.13.1 Introduction 35513.2 Target Selection: Identification of the Most Promising RNA Intervention Points 35713.3 Development of Robust Biophysical Methods, Alternative Strategies for Target Engagement, and Accurate and Reliable Functional Models 35813.3.1 Biophysical Methods for Interrogating Small Molecule–RNA Interactions 35813.3.2 Cellular Target Engagement Methods 36013.3.3 Unique Challenges Faced in the Development of Functional Assays for Studying Small Molecule–RNA Interactions 36413.4 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 36713.4.1 RNA Structure Prediction 36713.4.2 Computational Tools for Hit Optimization 36913.4.3 Implementation of Molecular Dynamics Simulations, Machine Learning, and AI Tools to Interrogate RNA–Small Molecule Interactions 37113.5 Deposition of Small Molecule–RNA Interaction Data with Rigorous Experimental Protocols and Controls is Needed 37313.6 Outlook: The Future of Small Molecule-Based RNA Therapeutics is Bright 375References 376Index 385

John Schneekloth, Jr., PhD (Jay), obtained his PhD from the chemistry department of Yale University. Following postdoctoral work at Princeton, Dr. Schneekloth joined the NCI, where his research involves using synthetic chemistry and high throughput chemical biology approaches to develop chemical probes of RNA, with a particular emphasis on targeting RNA with druglike small molecules.Martin Pettersson, PhD, obtained his PhD in Organic Chemistry from the University of Texas at Austin. He is a medical chemistry / drug discovery leader with over 20 years of industrial experience, mainly with Pfizer (co-inventor of Paxlovid). He has then continued as Senior Director of Neuroscience/Pain, Chemistry, at Grünenthal Boston Innovation Hub, and most recently as VP of Drug Discovery at Promedigen in South Korea.

• Series Editors’ Preface xiiiPreface xv1 Introduction 1John Schneekloth Jr. and Martin PetterssonReferences 42 RNA Structure Probing, Dynamics, and Folding 7Danny Incarnato2.1 Introduction 72.1.1 Relevance of RNA Structure in Disease 82.1.2 Challenges in Studying RNA Structures 82.2 Experimentally Guided RNA Structure Modeling 92.2.1 Structural Interrogation of RNA Nucleotides via Chemical Probing 102.2.1.1 Limits of RNA Chemical Probing 122.2.2 Direct Mapping of RNA–RNA Interactions 142.2.2.1 Limits of RNA–RNA Interaction Mapping 162.2.3 Mapping Spatially Proximal Nucleotides in RNA molecules 172.2.3.1 Limits of Methods for Spatial Proximity Mapping 172.3 Dealing with RNA Structure Heterogeneity 192.4 Querying RNA–Small Molecule Interactions with Chemical Probing 222.5 Conclusions and Future Prospects 22References 233 High-Resolution Structures of RNA 29Lukas Braun, Zahra Alirezaeizanjani, Roberta Tesch, and Hamed Kooshapur3.1 Introduction 293.2 X-Ray Crystallography 313.3 NMR Spectroscopy 343.4 Cryo-EM 373.5 3D Structure Prediction and Integrative Approaches 393.6 Conclusions 43Acknowledgments 43Conflicts of Interest 43References 434 Screening and Lead Generation Techniques for RNA Binders 49Gary Frey, Emily Garcia Sega, and Neil Lajkiewicz4.1 Knowledge-Based Versus Agnostic Screening 494.2 Virtual Screening 504.3 Screening Methods 514.3.1 High-Throughput Screening (HTS) 514.3.1.1 Mass Spectrometry 514.3.1.2 HTS of RNA Using Direct MS Approaches 524.3.1.3 HTS of RNA Using Indirect MS Approaches 544.3.1.4 DNA-Encoded Libraries (DELs) 564.3.1.5 Microarray Screening 574.3.1.6 Fragment-Based Drug Discovery 584.3.1.7 Phage Display 634.3.2 Orthogonal Methods 634.3.2.1 Surface Plasmon Resonance 634.3.2.2 Fluorescence-Based Assays 664.3.2.3 Microscale Thermophoresis (MST) 704.3.2.4 Isothermal Titration Calorimetry (ITC) 704.4 Binding Site Identification/Target Engagement 724.4.1 Covalent Methods 724.4.2 Competition with an Antisense Oligonucleotide (ASO) 744.5 Defining SAR and Functional Assays 754.5.1 Functional Assays 754.5.2 Phenotypic Screens 764.6 Identifying a Lead Series 764.6.1 Hit Optimization 774.6.2 Risdiplam Hit-to-Lead 784.6.3 Branaplam Lead Generation 794.6.4 Zotatifin Lead Generation 804.7 Concluding Thoughts and Outlook 80Acknowledgments 81References 815 Chemical Matter That Binds RNA 93Emily G. Swanson Hay, Zhengguo Cai, and Amanda E. Hargrove5.1 Introduction 935.2 Natural Ligands 945.2.1 Aminoglycosides 945.2.2 Tetracyclines 955.2.3 Macrolides 965.2.4 Native Riboswitch Ligands 965.3 Commercial Ligands 975.3.1 Industrial Libraries 985.3.2 Academic Libraries 985.4 Synthetic Ligands 995.4.1 Benzimidazoles and Purines 1005.4.2 Naphthalenes, Quinolines, and Quinazolines 1015.4.3 Oxazolidinones 1025.4.4 Amilorides 1025.4.5 Diphenyl Furan 1035.4.6 Multivalent Ligands 1035.5 Computational Tools for the Exploration of Chemical Space 1035.5.1 Similarity Searches and Principal Component Analysis 1045.5.2 Additional Machine-Learning Tools 1055.5.3 Structure-Based Ligand Design 1065.6 Case Studies in Examining and Expanding RNA-Targeted Chemical Space 1065.6.1 Using QSAR to Probe RNA-Targeting Small-Molecule Properties 1075.6.2 Evaluating the Chemical Space of Natural, Synthetic, and Commercial Ligands 1085.7 Conclusions and Outlook 111Acknowledgments 111References 1116 MicroRNAs as Targets for Small-Molecule Binders 119Maria Duca6.1 Introduction 1196.2 MicroRNAs 1216.3 MicroRNAs Biogenesis 1226.4 Targeting MicroRNAs with Small-Molecule RNA Binders 1236.4.1 Induction of miRNAs Expression: Tackling the Decrease of Tumor Suppressor miRNAs 1246.4.2 Inhibition of miRNAs Production: Pre- and Pri-miRNA Binders 1256.4.2.1 Discovery of miRNAs Inhibitors by Intracellular Assays 1256.4.2.2 Target-Based In Vitro Assays 1276.4.2.3 Design of Specific Ligands of Pre- and Pri-miRNAs 1316.4.2.4 Fragment-Based Drug Design 1386.4.2.5 DNA-Encoded Libraries (DELs) 1396.5 Inhibition of RNA–Protein Interactions in miRNAs Pathways 1406.6 Adding Cleavage Properties to miRNAs Interfering Agents 1426.7 Conclusions 144References 1447 Pre-mRNA Splicing Modulation 151Scott J. Barraza and Matthew G. Woll7.1 Introduction 1517.2 Overview of Splicing Biology 1527.2.1 The Spliceosome 1527.2.2 Classes of Alternative Splicing 1547.3 Pharmacological Mechanisms of Splicing Modulation 1557.3.1 Cis- and Trans-Regulatory Elements (Splicing Factors) 1557.3.1.1 Stabilization of Cis-Regulatory Elements 1567.3.1.2 Destabilization of Cis-Regulatory Elements 1587.3.1.3 Inhibition of Cis-Regulatory RNA–Protein Interactions 1587.3.1.4 Inhibition of Trans-Regulatory Elements 1607.3.1.5 Degradation of Trans-Regulatory Elements 1617.3.1.6 Inhibition of Trans-Regulatory Element Protein–Protein Interactions (PPIs) 1627.3.1.7 Stabilization of Trans-Regulatory Element RNA–Protein Interactions (RPIs) 1657.3.2 Kinases and Phosphatases 1657.3.2.1 Challenges in Targeting Kinases 1677.3.2.2 Inhibition of Kinases 1687.3.2.3 Activation and Degradation of Kinases 1687.3.2.4 Inhibition and Activation of Protein Phosphatases 1697.3.3 Epigenetic Writers and Erasers 1727.3.3.1 Inhibition of Epigenetic Writers 1727.3.4 RNA Helicases 1747.3.5 Drugging the Spliceosome 1757.3.5.1 Inhibition of U2 snRNP Recognition of the 3′-Splice Site 1767.3.5.2 E7107 1767.3.5.3 H3B-8800 1777.3.5.4 Stabilizers of U1 snRNP Recognition of the 5′-Splice Site 1807.3.5.5 Introduction to Spinal Muscular Atrophy (SMA) 1807.3.5.6 Risdiplam (Evrysdi®) 1837.4 Future Outlook 186References 1888 Prospects for Riboswitches in Drug Development 203Michael G. Mohsen and Ronald R. Breaker8.1 Introduction 2038.1.1 The Known Landscape of Riboswitches 2038.1.2 Riboswitches in Drug Development 2038.1.3 The Need for Novel Antibiotics 2058.2 Riboswitches as Drug Targets 2078.2.1 Why Target Riboswitches? 2078.2.2 Features of a Druggable Riboswitch 2088.2.3 Riboswitch-Targeted Drugs 2088.2.3.1 Small Molecules Targeting FMN Riboswitches 2088.2.3.2 Other Riboswitches Targeted in Proof-of-Principle Demonstrations 2098.2.4 Barriers and Future Developments 2108.3 Riboswitches as Tools for Antibiotic Drug Development 2108.3.1 Riboswitches as Biosensors 2108.3.2 A Riboswitch-Based Fluoride Sensor Illuminates Agonists of Fluoride Toxicity 2118.3.3 A Riboswitch-Based ZTP Sensor Identifies Inhibitors of Folate Biosynthesis 2118.3.4 A Riboswitch-Based SAH Sensor Reveals an Inhibitor of SAH Nucleosidase 2128.3.5 Barriers and Future Developments 2138.4 Application of Riboswitches in Gene Therapy 2138.4.1 Considerations for Designer Riboswitches 2138.4.2 Eukaryotic Expression Platforms 2148.4.3 Barriers and Future Developments 2168.5 Concluding Remarks 217Acknowledgment 218References 2189 Small Molecules That Degrade RNA 227Noah A. Springer, Samantha M. Meyer, Amirhossein Taghavi, Jessica L. Childs-Disney, and Matthew D. Disney9.1 Antisense Oligonucleotide Degraders 2279.2 Small-Molecule Direct Degraders 2289.2.1 N-Hydroxypyridine-2(1H)-thione (N-HPT) Conjugates 2299.2.2 Bleomycin 2299.2.3 Bleomycin Conjugates 2319.2.3.1 Bleomycin Degraders Targeting the r(CUG) Repeat Expansion That Causes DM1 2319.2.3.2 Bleomycin Degraders Targeting r(CCUG) Repeat Expansion that Causes DM2 2339.2.3.3 Bleomycin Degraders Targeting Oncogenic Precursor microRNAs 2339.2.3.4 Conclusions and Outlook for Bleomycin-Based Direct Degraders 2349.3 Ribonuclease Targeting Chimeras (RiboTACs) 2359.3.1 RNase L is an Endogenous Endoribonuclease That Functions as Part of the Innate Immune Response 2369.3.2 First-Generation RiboTACs Targeting Oncogenic miRNAs 2369.3.3 Small-Molecule-Based RiboTACs 2399.3.4 Comparison of Bleomycin-Based Direct Degraders and RiboTACs 2429.3.5 Discovery of Additional Small-Molecule RNase L Activators 2429.3.6 Conclusions and Outlook for RiboTACs 2439.4 Summary and Outlook for Small-Molecule RNA Degraders 244References 24610 Approaches to the Identification of Molecules Altering Programmed Ribosomal Frameshifting in Viruses 253Elinore A. VanGraafeiland, Diego M. Arévalo, and Benjamin L. Miller10.1 Introduction 25310.2 Mechanisms of Frameshifting 25610.3 Targeting Frameshifting in HIV 25710.4 Targeting Frameshifting in SARS-CoV-1 and SARS-CoV-2 26310.5 Conclusions 274References 27411 RNA–Protein Interactions: A New Approach for Drugging RNA Biology 281Dalia M. Soueid and Amanda L. Garner11.1 Molecular Basis of RNA–Protein Interactions 28211.1.1 RNA Recognition Motifs (RRMs) 28211.1.2 Double-Stranded RNA-Binding Domains (dsRBD) 28611.1.3 Zinc Finger (ZnF) Domains 28711.1.4 K Homology (KH) Domains 28911.1.5 Other RBDs 29011.2 Regulation and Dysregulation of RNA–Protein Interactions 29011.2.1 Poor Quality Control Leads to Over- and Underproduction of RBPs 29211.2.2 RBPs Become Out of Control, mRNA Processing Gets a Makeover (and Hates It) 29411.2.3 RBP Shuttling of mRNA Becomes Askew 29411.2.4 The RBP is Lost and Wreaks Havoc on the Cell 29511.2.5 RBPs Dictate Which mRNAs are Translated, Favoring their Toxic Friends 29511.2.6 RBPs and RNA Become Very Clique-y, Form Their Own Complex and Cause Stress to the Rest of the Cell 29611.3 Experimental Methods to Detect and Screen for Small Molecules that Modulate RNA–Protein Interactions 29711.3.1 In vitro Fluorescence-Based Assays 29711.3.2 In vitro Chemiluminescence-Based Assays 29711.3.2.1 Cell-Based RPI Detection Assays 30011.3.3 Cell-Based RNA–Protein Interaction Screening 30111.4 Closing Remarks 302References 30312 Drugging the Epitranscriptome 321Tanner W. Eggert and Ralph E. Kleiner12.1 Introduction 32112.2 Modifications on mRNA: N6-Methyladenosine, Pseudouridine, and Inosine 32512.2.1 N6-Methyladenosine (m 6 A) 32512.2.2 Pseudouridine (Ψ) 32712.2.3 Inosine (I) 32812.3 Modifications on tRNA and rRNA 33012.3.1 tRNA Modifications 33012.3.2 rRNA Modifications 33412.4 Concluding Remarks 335References 33613 Outlook 355Christopher R. Fullenkamp, Xiao Liang, Martin Pettersson, and John Schneekloth Jr.13.1 Introduction 35513.2 Target Selection: Identification of the Most Promising RNA Intervention Points 35713.3 Development of Robust Biophysical Methods, Alternative Strategies for Target Engagement, and Accurate and Reliable Functional Models 35813.3.1 Biophysical Methods for Interrogating Small Molecule–RNA Interactions 35813.3.2 Cellular Target Engagement Methods 36013.3.3 Unique Challenges Faced in the Development of Functional Assays for Studying Small Molecule–RNA Interactions 36413.4 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 36713.4.1 RNA Structure Prediction 36713.4.2 Computational Tools for Hit Optimization 36913.4.3 Implementation of Molecular Dynamics Simulations, Machine Learning, and AI Tools to Interrogate RNA–Small Molecule Interactions 37113.5 Deposition of Small Molecule–RNA Interaction Data with Rigorous Experimental Protocols and Controls is Needed 37313.6 Outlook: The Future of Small Molecule-Based RNA Therapeutics is Bright 375References 376Index 385