Protein Degradation with New Chemical Modalities: Successful Strategies in Drug Discovery and Chemical Biology (Issn)

Targeting protein degradation using small molecules is one of the most exciting small-molecule therapeutic strategies in decades and a rapidly growing area of research. In particular, the development of proteolysis targeting chimera (PROTACs) as potential drugs capable of recruiting target proteins to the cellular quality control machinery for elimination has opened new avenues to address traditionally ‘difficult to target’ proteins. This book provides a comprehensive overview from the leading academic and industrial experts on recent developments, scope and limitations in this dynamically growing research area; an ideal reference work for researchers in drug discovery and chemical biology as well as advanced students. Cover Half-title Series information Title page Copyright information Preface Table of contents Chapter 1 PROTAC-mediated Target Degradation: A Paradigm Changer in Drug Discovery? References Chapter 2 Structural and Biophysical Principles of Degrader Ternary Complexes 2.1 Introduction 2.1.1 Mechanistic Advantages of Targeted Protein Degradation 2.1.1.1 Immediate Advantages of Degradation Versus Inhibition 2.1.1.2 Differentiation of Degraders due to Their Mode of Action 2.1.2 History of PROTACs (2001–2010) 2.1.3 Small-molecule VHL- and CRBN-based PROTACs (2010–2015) 2.2 Structural Features of Ternary Complexes 2.2.1 Ternary Complex Equilibria and Definitions 2.2.2 Structural Elucidation of PROTAC Ternary Complexes 2.2.2.1 The First PROTAC Ternary Complex Crystal Structure: VHL:MZ1:Brd4BD2 2.2.2.2 Structure-guided design of SMARCA2/4 PROTACs 2.2.2.3 Ternary Structures of CRBN-based PROTACs 2.2.3 Degraders as Monovalent Molecular Glues 2.2.3.1 Cereblon-targeting Immunomodulatory Drugs 2.2.3.2 DCAF15-targeting Sulfonamide Drugs 2.2.4 Surface Areas Buried by PROTACs and Monovalent Glues 2.3 Ternary Assays 2.3.1 Can My PROTAC Form a Ternary Complex? 2.3.1.1 Pull-down Assays 2.3.1.2 Proximity-based Ternary Assays: AlphaScreen/LISA and TR-FRET 2.3.1.3 Surface Plasmon Resonance 2.3.2 How Tightly Does My Ternary Complex Bind? 2.3.2.1 Competition Assays 2.3.2.2 Direct Binding Assays 2.3.3 To What Extent Is My Ternary Complex Cooperative? 2.3.4 How Long Does My Ternary Complex Last? 2.3.5 Does the PROTAC Induce Ternary Complex Formation in Cells? 2.3.5.1 Separation of Phases-based Protein Interaction Reporter Assay (SPPIER) 2.3.5.2 Bioluminescence Resonance Energy Transfer (BRET) 2.4 Concluding Remarks 2.5 Acknowledgments 2.5.1 Funding 2.5.2 Conflict of Interest Statement References Chapter 3 Immediate and Selective Control of Protein Abundance Using the dTAG System 3.1 The Potential and Limitations of Targeted Protein Degradation 3.2 Chemical–Genetic Degradation Approaches 3.3 Development of the dTAG Platform 3.4 Genetic Methods to Express FKBP12F36V-fusions 3.4.1 Ectopic Expression of FKBP12F36V-fusions 3.4.2 Knock-in Strategies to Express FKBP12F36V-fusions 3.5 Strategies Towards Identification of a Lead dTAG Molecule 3.5.1 Biochemical Assays for FKBP12F36V and E3 Ligase Binding 3.5.2 Determining FKBP12F36V-specific Degradation in Cells 3.5.3 Requirement of E3 Ligase and Proteasome 3.5.4 Assessment of dTAG Molecule Selectivity 3.5.5 In Vivo Assessment of dTAG Molecule Activity 3.6 Case Studies Employing the dTAG Platform 3.6.1 Target Validation Using dTAG 3.6.2 Targeting Recalcitrant Oncoproteins Using dTAG 3.6.3 Targeting Essential Transcriptional Regulators Using dTAG 3.7 General Considerations for Employing Tag-based Strategies 3.8 Concluding Remarks 3.9 Abbreviations 3.10 Acknowledgments 3.10.1 Competing Financial Interests References Chapter 4 Developing Pharmacokinetic/Pharmacodynamic Relationships With PROTACs 4.1 Introduction 4.2 The Importance of PK/PD Relationships and Additional Considerations for PROTACs 4.2.1 Building PK/PD Relationships 4.2.2 PK/PD Considerations for PROTACs 4.2.2.1 Catalytic Mechanism of PROTACs 4.2.2.2 Impact of Protein Half-life 4.2.2.3 Rate of PROTAC-mediated Degradation 4.2.2.4 Functional Consequences of PROTAC Binding to the Degradation Target 4.2.2.5 E3 Ligase and Target Protein Distribution 4.3 Developing PK/PD Relationships for a Series of RIPK2 PROTACs 4.3.1 Design of PK/PD Experiments for RIPK2 PROTACs 4.3.2 Results from PK/PD Experiments with RIPK2 PROTACs 4.4 PBPK/PD Models for PROTACs 4.5 Conclusions 4.6 Ethical Review References Chapter 5 New Activities of CELMoDs, Cereblon E3 Ligase-modulating Drugs 5.1 Introduction 5.2 Targeted Protein Degradation Through Cereblon-CRL4 5.3 CRL4 Architecture 5.4 CELMoD Mechanism of Action 5.5 Identification of CELMoDs with Novel Activities 5.6 Molecular Basis for Substrate Recruitment 5.7 Identification of a Substrate Mediating Teratogenicity Through a Structural Degron Search 5.8 Expansion of Cereblon Neosubstrates 5.9 Further CELMoDs in Clinical Development 5.9.1 Avadomide 5.9.2 Iberdomide 5.9.3 CC-90009 and CC-92480 5.10 The Development of Cereblon-targeting Hetero-bifunctional Degraders 5.11 Differences Between Hetero-bifunctional and Scaffolded Protein–Protein Interaction Ubiquitin Ligase Modulators 5.12 Conclusions References Chapter 6 Structure-based PROTAC Design 6.1 Introduction 6.2 PROTAC Design – Differences to Small Molecules 6.3 Structure-based Linkerology 6.4 Learning from PPI Stabilization 6.5 VHL PROTAC Ternary Complexes 6.6 Cereblon PROTAC Ternary Complexes 6.7 Identifying the Right PPI to Target 6.8 Future Technologies for Structure-based PROTAC Design 6.9 Acknowledgments References Chapter 7 Plate-based High-throughput Cellular Degradation Assays to Identify PROTAC Molecules and Protein Degraders 7.1 Introduction 7.2 PROTACs 7.3 Plate-based Assays to Measure Protein Degradation 7.3.1 Immunofluorescent Target Imaging 7.3.2 ELISA or Derivative Assay Technologies 7.3.3 Protein Tagging 7.3.4 Validation of Assays to Measure Protein Degradation 7.4 Plate-based Assays to Understand Degrader Mechanism – PROTACS 7.4.1 Cellular Permeability and Target Engagement Assays 7.4.2 PROTAC Ternary Complex Formation 7.4.3 PROTAC-mediated Ubiquitination 7.4.4 Proteasome Recognition Assays 7.4.5 Modification of Degradation Assays to Assess POI Abundance and Turnover 7.4.6 Mechanistic Tools References Chapter 8 PROTAC Targeting BTK for the Treatment of Ibrutinib-resistant B-cell Malignancies 8.1 Introduction 8.2 Review of BTK Inhibitors 8.3 Drug Resistance and Side Effects of Ibrutinib 8.4 PROTAC Contributes to Overcome Drug Resistance of Ibrutinib 8.5 Summary 8.6 Acknowledgments References Chapter 9 An Efficient Approach Toward Drugging Undruggable Targets 9.1 Introduction 9.2 Target Identification and Validation 9.2.1 Use of AID Technology In Vivo 9.3 Efficient Drug Discovery Platform, RaPPIDS 9.3.1 Proprietary E3 Ligase Binders 9.3.2 A Strategy for Drug Candidate Discovery by RaPPIDS Platform 9.4 Case Study of RaPPIDS for 1st Program, IRAK-M Degrader 9.4.1 Background of IRAK-M 9.4.2 Drug Discovery of IRAK-M Degrader by RaPPIDS 9.5 Future Perspectives 9.6 Conclusion 9.7 Abbreviations 9.8 Acknowledgments References Chapter 10 E3-mediated Ubiquitin and Ubiquitin-like Protein Ligation: Mechanisms and Chemical Probes 10.1 Introduction 10.2 E3-dependent Conformational Regulation of E2~UB Intermediates 10.3 UBL Transfer to Substrates by Adaptor E3s Harboring RING and RING-like Domains 10.3.1 Cullin-RING Ligases (CRL) 10.3.2 CRL-dependent Ubiquitylation 10.3.3 CRL Modification by NEDD8 10.3.4 Small Molecules Manipulating Cullin Neddylation 10.3.5 CRL Assembly Cycle 10.3.6 RING-between-RING (RBR) Ligases 10.3.7 Unique Domains Specify Activation and Activity of RBR E3s 10.3.8 HECT E3 Ligases 10.3.9 Catalytic HECT Domain 10.3.10 HECT E3-mediated Polyubiquitylation 10.3.11 Modulation of NEDD4-family HECT E3 Activity by UB Binding to an N-lobe Exosite 10.3.12 HECT Domain Oligomerization 10.3.13 Mechanisms to Regulate HECT E3 Ubiquitylation Activity 10.4 Cysteine-reactive Probes 10.5 Chemical Biology Approach with Reactive E2~UB Conjugates Reveal RING-Cys-relay (RCR) Ligase 10.6 Conclusions and Future Perspectives Note added on proof References Chapter 11 Plant E3 Ligases as Versatile Tools for Novel Drug Development and Plant Bioengineering 11.1 Introduction 11.2 The Four Classes of E3 Ligases in Higher Plants: A Brief Overview 11.2.1 Monomeric E3 RING-finger Ligases 11.2.2 Cullin-based RING E3 Ligases 11.2.3 U-box E3 Ligases 11.2.4 HECT E3 Ligases 11.3 Pathogens’ Use of the Ubiquitin Proteasome Pathway 11.4 The Ubiquitin Proteasome Pathway as an Opportunity 11.4.1 Novel Drug Development Utilizing the UPP 11.4.2 Synthetic Biology Using UPP Sensors 11.4.3 The N-degron Pathway as Bioengineering Tool 11.5 Future Perspectives 11.6 Acknowledgments References Chapter 12 Deubiquitinase Inhibitors: An Emerging Therapeutic Class 12.1 Introduction/Background 12.2 Biology and Clinical Opportunity for DUB Inhibition 12.2.1 USP7 12.2.2 USP22 12.2.3 OTULIN 12.2.4 USP30 12.3 Validating Inhibitors and Substrates of DUBs 12.3.1 Substrate Validation 12.3.2 Inhibitor Validation 12.4 Examples of Inhibitors 12.4.1 USP25/28 12.4.2 CSN5 12.4.3 Rpn11 12.4.4 USP7 12.5 Outlook and Future Directions References Chapter 13 Targeting Translation Regulation for the Development of Novel Drugs 13.1 Introduction 13.2 PSM, Discovery of Translation Regulators Using Pairs of Fluorescent tRNAs 13.3 Target Space for PSM: From Transcription to Translation 13.3.1 RNA Processing 13.3.2 RNA-binding Proteins 13.3.3 mRNA Localization 13.3.4 mRNA Translation 13.3.5 tRNA Modifications, Expression and Aminoacylation References Chapter 14 Classes, Modes of Action and Selection of New Modalities in Drug Discovery 14.1 Introduction 14.2 Nucleic Acid-based Modalities 14.2.1 Targetable Modes of Action 14.2.1.1 Protein Recognition 14.2.1.2 Direct and Indirect Downregulation of RNA Levels 14.2.1.3 Direct and Indirect Upregulation of RNA Levels 14.2.1.4 Genome Editing 14.2.2 Classes of Nucleic Acid-based Modalities 14.2.2.1 Antisense Oligonucleotide (ASO) and Small Interfering RNA (siRNA) 14.2.2.1.1 Chemical Modifications. 14.2.2.1.2 Design. 14.2.2.2 Modified mRNA (modRNA) 14.2.2.3 Aptamers 14.2.3 Strengths and Limitations of Nucleic Acid-based Modalities 14.2.3.1 Delivery 14.2.3.2 Tissue Distribution 14.2.3.3 Safety 14.3 Hyper-modified Peptides 14.3.1 Targetable Modes of Action 14.3.2 Classes of Hyper-modified Peptidic Modalities 14.3.2.1 Monocyclic Peptides Including Stapled Peptides and Other Protein Structure Mimetics 14.3.2.2 Polycyclic Peptides 14.3.3 Strength and Limitations of Peptide-based Modalities 14.4 Hybrid and Multivalent Modalities 14.4.1 Modality Fusion for Synergistic Binding and Polypharmacology 14.4.2 Modality Conjugation for Synergistic Binding 14.4.3 Enablement of Novel MOAs with Hybrid Modalities 14.4.4 Strength and Limitations of Hybrid Modalities 14.5 Selection of Modalities in Drug Discovery 14.5.1 Repertoire of Modes of Action and Modalities 14.5.1.1 Targeting at the DNA Level 14.5.1.2 Targeting at the RNA Level 14.5.1.3 Targeting at the Protein Level 14.5.2 Criteria and Perspective for Selecting Modalities 14.6 Conclusion References Chapter 15 Small-molecule Targeted Degradation of RNA 15.1 Introduction 15.2 Design Strategy for RNA Cleavers and Degraders 15.3 Cleaving r(CUG)exp via Photolysis of N-hydroxy-2(1H)-thione (HPT) 15.4 Harnessing the Cleavage Potential of the Bleomycin Family of Natural Products 15.4.1 Targeted Cleavage of r(CUG)exp 15.4.2 Targeted Cleavage of pri-miR-96 15.5 Harnessing the Potential of RNA Cellular Degradation Machinery 15.5.1 Recruitment of RNase L for Targeted Degradation of miR-96 15.5.2 Recruitment of RNase L for Targeted Degradation of miR-210 15.6 Outlook and Conclusions References Index