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

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Half-title
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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