Cover
List of Contributors
Preface
Part I: Overview, Concepts, and Approaches
1. 1 The Evolving Role of Structural Biology in Drug Discovery
  1. 1.1 Introduction
  2. 1.2 The Expanding Toolbox of Structural Biology for Drug Discovery
  3. 1.3 The Various Uses of Structural Biology in Drug Discovery
  4. 1.4 Evolving Drugs and Targets
  5. 1.5 Current Trends and Perspectives
  6. References
2. 2 A Structural View on Druggability: Experimental and Computational Approaches
  1. 2.1 Introduction
  2. 2.2 Views on Target Druggability
  3. 2.3 In Silico Methods for Druggability Assessment of Targets with Well‐defined Pockets
  4. 2.4 Experimental Methods for Druggability Assessment
  5. 2.5 A Challenge for Druggability Predictions: Protein–Protein Interactions
  6. 2.6 Perspective
  7. References
3. 3 Structural Chemogenomics: Profiling Protein–Ligand Interactions in Polypharmacological Space
  1. 3.1 Introduction
  2. 3.2 Simultaneously Targeting Multiple Proteins Can Be More Efficient in Disrupting Disease Mechanism
  3. 3.3 Computer‐Aided Approaches for Profiling Bioactivities of Ligands
  4. 3.4 Applications
  5. 3.5 Conclusion
  6. References
4. 4 Fragment‐Based Ligand Discovery
  1. 4.1 Introduction
  2. 4.2 The Evolution of FBLD
  3. 4.3 The FBLD Process
  4. 4.4 Fragment Libraries
  5. 4.5 Maintaining a Fragment Library
  6. 4.6 Fragment Screening
  7. 4.7 Integrating Fragments with Other Compounds
  8. 4.8 Validating Fragment Hits: Comparing Methods
  9. 4.9 Fragment Hit Rates
  10. 4.10 Determining Structures of Fragment: Protein Complexes
  11. 4.11 Fragment Evolution
  12. 4.12 Concluding Remarks
  13. Acknowledgments
  14. References
5. 5 Combining Structural, Thermodynamic, and Kinetic Information to Drive Hit‐to‐Lead Progression
  1. 5.1 Introduction
  2. 5.2 The Role of Thermodynamics in Hit to Lead
  3. 5.3 The Role of Kinetics in Hit to Lead
  4. 5.4 Summary
  5. References
6. 6 Allostery as Structure‐Encoded Collective Dynamics: Significance in Drug Design
  1. 6.1 Introduction
  2. 6.2 Computational Methods
  3. 6.3 Applications
  4. 6.4 Future Directions and Conclusion
  5. References
Part II: Tool
1. 7 Biophysical Assessment of Target Protein Quality in Structure‐Based Drug Discovery
  1. 7.1 Biophysical Methods in Drug Discovery
  2. 7.2 Case Study I: Micro‐Inhomogeneity
  3. 7.3 The Role of Biophysical Methods in the Optimization of Protein Crystallization
  4. 7.4 Case Study II: Minimizing Macro‐Inhomogeneity
  5. 7.5 Outlook and Concluding Remarks: Requirements for Upcoming Biophysical Methods
  6. Acknowledgment
  7. References
2. 8 An Industrial Perspective on Protein–Ligand Complex Crystallization
  1. 8.1 Introduction to Co‐crystal Structures and Drug Development
  2. 8.2 Basics of X‐Ray Analysis of Co‐crystals
  3. 8.3 Ligands
  4. 8.4 Preparing Protein for Successful Crystallization
  5. 8.5 Crystallization
  6. 8.6 Selecting Ligands for Crystallization
  7. 8.7 Methods for Obtaining Co‐Crystals: Soaking and Co‐crystallization
  8. 8.8 Future Perspective
  9. Acknowledgment
  10. References
3. 9 Membrane Protein Crystallization
  1. 9.1 Introduction
  2. 9.2 Membrane Protein Production
  3. 9.3 Amphipathic Manipulation of Membrane Proteins
  4. 9.4 Preparing Protein Samples for Crystallization
  5. 9.5 Membrane Protein Crystallization
  6. 9.6 Methods for Determining Membrane Protein Structures
  7. 9.7 Conclusion and Outlooks
  8. References
4. 10 High‐Throughput Macromolecular Crystallography in Drug Discovery: Evolving in the Midst of Revolutions
  1. 10.1 Introduction
  2. 10.2 Setting the Scene for Evolution and Revolutions
  3. 10.3 Baseline: The “Human, All Too Human” Workflow of Early MX for SBDD
  4. 10.4 First Wave of Automation Toward High‐Throughput Operation: Robotics Without Refactoring
  5. 10.5 Second Wave of Automation: The Twin Tracks of In Situ Crystallography and Microcrystallography
  6. 10.6 An Emerging Third Wave of Automation: Serial Microcrystallography
  7. 10.7 From Diffraction Data to Structural Results: Evolving Best Practices
  8. 10.8 Conclusions and Outlook: Whither HTMX for Drug Discovery?
  9. Acknowledgments
  10. References
5. 11 Assessment of Crystallographic Structure Quality and Protein–Ligand Complex Structure Validation
  1. 11.1 Introduction
  2. 11.2 Quality Parameters
  3. 11.3 Dataset Quality
  4. 11.4 Low‐Resolution Structures
  5. 11.5 Possible Influence of Crystal Packing
  6. 11.6 Software Tools
  7. 11.7 Analysis of Quality Metrics
  8. 11.8 Conclusions
  9. Acknowledgments
  10. References
6. 12 Complementary Information from Neutron Crystallography Studies
  1. 12.1 Introduction
  2. 12.2 Differences in Characteristics of X‐Rays and Neutrons
  3. 12.3 A Brief History of Protein Neutron Crystallography
  4. 12.4 Facility, Neutron Source, and Detector
  5. 12.5 Current Status of Neutron Protein Crystallography
  6. 12.6 Method for Neutron Crystallography of Proteins
  7. 12.7 General Information Obtained from Neutron Crystallography
  8. 12.8 Use of Neutron Crystallography for the Structure Analysis of Protein Drug Targets
  9. 12.9 Use of Neutron Protein Crystallography for Drug Design
  10. 12.10 Future Perspectives of the Use of Neutron Crystallography for Drug Design
  11. References
7. 13 Determination of Protein Structure and Dynamics by NMR: State of the Art and Application to the Characterization of Biotherapeutics
  1. 13.1 Introduction
  2. 13.2 Solution Structure Determination of Macromolecules by NMR
  3. 13.3 Assignment and Labeling Strategies
  4. 13.4 NMR and Dynamic Aspects
  5. 13.5 Biomolecular Dynamics by NMR
  6. 13.6 Intrinsically Disordered Proteins
  7. 13.7 Alternative Approaches for Non‐soluble Proteins
  8. 13.8 Optimized Strategies for the Study of Biomolecules in Solution
  9. 13.9 Conclusion
  10. References
8. 14 NMR Studies of Protein–Small Molecule Interactions for Drug Discovery
  1. 14.1 Introduction
  2. 14.2 Early‐Stage Discovery
  3. 14.3 Lead Optimization
  4. 14.4 Emerging Applications/Fields
  5. 14.5 Outlook
  6. References
9. 15 Computational Structural Biology for Drug Discovery: Power and Limitations
  1. 15.1 Introduction
  2. 15.2 Converting PDB Entries into Full‐Atom Models
  3. 15.5 Future Perspective
  4. Acknowledgments
  5. References
Part III: Structure-Based Discovery in Some Important orPromising Targets and Therapeutic Families
1. 16 The Role of Structural Biology in Kinase Inhibitor Drug Discovery Success
  1. 16.1 Protein Kinases and Their Structural Elements: A Dynamic Landscape for Drug Discovery
  2. 16.2 The Challenge of Selectivity and Drug Resistance: Design and Discovery of Afatinib, the First Irreversible Protein Kinase Inhibitor Approved for Cancer Treatment
  3. 16.3 When Structural Biology Drives Chemistry to Therapeutic Breakthrough: The Vemurafenib Case History
  4. 16.4 Second‐Generation Anaplastic Lymphoma Kinase Inhibitors: The Discovery of the First‐in‐Class Drug Ceritinib
  5. 16.5 The Discovery of Type II Inhibitor Ponatinib: A Milestone in the Struggle Against the ABL Gatekeeper Resistant Mutation T315I
  6. 16.6 Protein Kinase Inhibitor Drug Discovery and Structural Biology: Future Perspective
  7. References
2. 17 Serine Proteinases from the Blood Coagulation Cascade
  1. 17.1 Introduction
  2. 17.2 Thrombin
  3. 17.3 Factor Xa (FXa)
  4. 17.4 Factor VIIa (FVIIa)
  5. 17.5 Further Developments
  6. 17.6 Factor IXa
  7. 17.7 Factor XIa
  8. 17.8 Impact of Structure‐Based Drug Design
  9. References
3. 18 Epigenetic Proteins as Emerging Drug Targets
  1. 18.1 Introduction
  2. 18.2 Acetylation/Deacetylation
  3. 18.3 Methylation/Demethylation
  4. 18.4 Other Epigenetic Modifications
  5. 18.5 The Future of Epigenetic Drug Discovery
  6. Acknowledgments
  7. References
4. 19 Impact of Recently Determined Crystallographic Structures of GPCRs on Drug Discovery
  1. 19.1 G Protein‐Coupled Receptors as Pharmaceutical Targets
  2. 19.2 Topology and Classes of GPCRs
  3. 19.3 GPCR X‐Ray Crystal Structures
  4. 19.4 Class A GPCR Small Ligand Binding Sites and Druggability
  5. 19.5 Class B GPCR X‐Ray Crystal Structures
  6. 19.6 Class C GPCRs and Allosteric Modulators
  7. 19.7 GPCR Activation
  8. 19.8 Structure‐Based Approaches to GPCR Drug Discovery
  9. 19.9 Conclusion
  10. References
5. 20 Targeting Protein–Protein Interactions Perspective
  1. 20.1 Introduction
  2. 20.2 Detection and Analysis of PPIs
  3. 20.3 PPI Screening
  4. 20.4 Examples
  5. 20.5 Stapled Peptides
  6. 20.6 Alternatives to Small‐Molecule Orthosteric Inhibition
  7. 20.7 Conclusion and Perspectives
  8. References
6. 21 Mass Spectrometry‐Based Strategies for Therapeutic Antibodies Extensive Characterization and Optimization (OptimAbs)
  1. 21.1 Introduction
  2. 21.2 Intact mAb Analysis
  3. 21.3 Middle‐Up mAb Analysis
  4. 21.4 Bottom‐Up Peptide Mapping for Primary Structure Assessment
  5. 21.5 Top‐Down Approaches for mAb Sequencing
  6. 21.6 Hydrogen/Deuterium Exchange Mass Spectrometry
  7. 21.7 Native MS and Ion Mobility–Mass Spectrometry (IM‐MS) for the Characterization of mAb/Ag Binding Stoichiometry and of Protein Conformation
  8. 21.8 From Optimized Antibodies (OptimAbs) to Optimized Antibody–Drug Conjugates (OptimADCs)
  9. 21.9 Concluding Remarks
  10. Acknowledgments
  11. References
Part IV: Challenges and New Frontiers
1. 22 Integrating Evolution of Drug Resistance into Drug Discovery: Lessons from the Viral Proteases of HIV‐1 and HCV
  1. 22.1 Evolution of Antiviral Drug Resistance
  2. 22.2 Substrate Envelope in Drug Design
  3. 22.3 Protein Dynamics Is Key to Molecular Recognition
  4. 22.4 Robust Drug Design: Hitting Multiple Targets at a Time
  5. 22.5 Future Perspective: Integrating Evolution and Conformational Dynamics into Drug Design
  6. Acknowledgments
  7. References
2. 23 A Comprehensive Review on Mycobacterium tuberculosis Targets and Drug Development from a Structural Perspective
  1. 23.1 Introduction
  2. 23.2 Tuberculosis
  3. 23.3 Mycobacterium tuberculosis
  4. 23.4 Tuberculosis Drug Discovery and Development
  5. 23.5 Conclusion and Perspectives
  6. References
3. 24 Using Crystal Structures of Drug‐Metabolizing Enzymes in Mechanism‐Based Modeling for Drug Design
  1. 24.1 Structure‐Based Modeling of Cytochrome P450s
  2. 24.2 Other Phase I Drug‐Metabolizing Enzymes
  3. 24.3 Phase II Drug‐Metabolizing Enzymes
  4. 24.4 Interplay Between Metabolism and Inhibition
  5. 24.5 Future Directions
  6. Acknowledgment
  7. References
4. 25 Intrinsically Disordered Proteins: Targets for the Future?
  1. 25.1 Introduction
  2. 25.2 IDPs as Novel Drug Targets
  3. 25.3 Molecular Mechanisms of Drugs Targeting Ordered Proteins
  4. 25.4 Disorder‐Based Rational Drug Design
  5. 25.5 Direct Targeting of IDPs/IDPRs
  6. 25.6 Targeting Functionally Misfolded IDPs: A Hypothesis
  7. 25.7 Targeting Intrinsically Disordered Structural Ensembles: α‐Synuclein as an Illustration
  8. 25.8 Targeting Aggregating IDPs
  9. 25.9 Conclusions
  10. Acknowledgments
  11. References
5. 26 Cryo‐electron Microscopy as a Tool for Drug Discoveryin the Context of Integrative Structural Biology
  1. 26.1 Introduction
  2. 26.2 The Resolution Revolution
  3. 26.3 What Is Cryo‐EM?
  4. 26.4 The Cryo‐EM Single‐Particle Analysis Workflow
  5. 26.5 Selected Case Studies
  6. 26.6 Summary and Conclusion
  7. References
6. 27 Application of Hard‐X‐Ray Free‐Electron Lasers for Static and Dynamic Processes in Structural Biology
  1. 27.1 Introduction: Overview of X‐Ray Free‐Electron Lasers
  2. 27.2 Comparison with Conventional X‐Ray Crystallography
  3. 27.3 XFEL Structures: Successes Since 2009
  4. 27.4 Challenges in XFELs
  5. 27.5 Future Outlook
  6. 27.6 Conclusion
  7. Acknowledgments
  8. References
Index
End User License Agreement

List of Tables

Chapter 1
1. Table 1.1 Direct imaging techniques used in drug discovery.
2. Table 1.2 Selected key events in the development of the three main techniques...
Chapter 2
1. Table 2.1 Overview of druggability assessment methods of targets with well‐de...
2. Table 2.2 Selected examples of druggable protein–protein interaction targets.
Chapter 5
1. Table 5.1 Different types of sensor chips available for SPR experiments.
2. Table 5.2 Parameters describing kinetics of association and dissociation for ...
Chapter 7
1. Table 7.1 Methods commonly and predominantly applied for the characterization...
2. Table 7.2 Compilation of biophysical methods used to characterize proteins.
3. Table 7.3 Average signal‐weighted sedimentation coefficients (N = 2) normaliz...
Chapter 12
1. Table 12.1 Neutron diffractometers for macromolecular crystallography.
Chapter 13
1. Table 13.1 Online resources for solution structure determination based on NMR...
2. Table 13.2 Programs for molecular modeling and structure validation.
3. Table 13.3 The different types of NMR instruments found in a structural biolo...
4. Table 13.4 The different types of protein samples amenable to NMR studies, al...
5. Table 13.5 The different types of NMR studies.
Chapter 16
1. Table 16.1 Protein kinase inhibitors approved by the Food and Drug Administra...
2. Table 16.2 Growth inhibition data on lung cancer cell lines reported as nM va...
Chapter 18
1. Table 18.1 Classes of epigenetic protein families.
Chapter 19
1. Table 19.1 List of published representative GPCR X‐ray crystal structures.
Chapter 21
1. Table 21.1 Theoretical and experimental masses obtained by middle‐up analyses...
Chapter 22
1. Table 22.1 Cleavage site sequences for HIV‐1 and HCV NS3/4A proteases.
2. Table 22.2 Reported sites of drug resistance mutations in HCV NS3/4A protease...
Chapter 23
1. Table 23.1 TB drugs in clinical use and their mechanism of action/target.
2. Table 23.2 Structural coverage of the proteome ofMtb H37Rv with respect to th...
3. Table 23.3 TB drugs recently approved or in development and their mechanism o...
Chapter 27
1. Table 27.1 Comparison of SFX and synchrotron data statistics.
2. Table 27.2 Comparison and status of various available XFELs.

List of Illustrations

Chapter 1
1. Figure 1.1 Various uses of structural biology in the drug discovery pipeline...
Chapter 2
1. Figure 2.1 General approach for druggability predictions based on structural...
2. Figure 2.2 Difficult targets with known drugs, a challenge for druggability ...
Chapter 3
1. Figure 3.1 A schematic depiction of a biological protein–protein (signaling ...
2. Figure 3.2 Different ways of adopting a multi‐target therapeutic approach. D...
3. Figure 3.3 The different computational methods for multi‐target drug discove...
4. Figure 3.4 A schematic depiction of the biological activity of a set of liga...
5. Figure 3.5 Schematic depiction of proteochemometric modeling. Ligand and pro...
Chapter 4
1. Figure 4.1 The schematic summarizes the steps in the SAR‐by‐NMR process: pro...
2. Figure 4.2 The fragment‐based drug discovery process at Vernalis.
3. Figure 4.3 Fragment library design. The different selection steps (A–E) are ...
4. Figure 4.4 Fragment screening. An oft‐quoted representation that shows the a...
5. Figure 4.5 Competitive ligand‐observed NMR spectroscopy.
6. Figure 4.6 A schematic representation of surface plasmon resonance (SPR).
7. Figure 4.7 Growing and merging fragments – (a) example of Aurora kinase. The...
Chapter 5
1. Figure 5.1 A typical thermogram from an iTC₂₀₀ instrument.
2. Figure 5.2 A typical sensorgram from a BIAcore instrument.
3. Figure 5.3 (a) Pocket changes on binding “type I” and “type II” kinase inhib...
Chapter 6
1. Figure 6.1 Schematic description of allosteric conformational changes in the...
2. Figure 6.2 Examination of the conformational space accessible to proteins by...
3. Figure 6.3 Results from druggability simulations. (a) Identification of four...
4. Figure 6.4 Diversification of cellular signaling pathways by allostery. GPCR...
Chapter 7
1. Figure 7.1 (a) Elution profile (280 nm, blue) for the preparative chromatogr...
2. Figure 7.2 (a) Determination of K _M for the substrate Z‐Phe‐Arg‐AMC was perfo...
3. Figure 7.3 (a) SEC elution profile of CPT‐2. The black horizontal bar at V _e ...
4. Figure 7.4 (a) AUC absorbance scans (280 nm) for the analysis of CPT‐2 with ...
Chapter 8
1. Figure 8.1 Schematic representation of the parallel processing approach in p...
2. Figure 8.2 Playground of parameters in protein–ligand crystallization. This ...
3. Figure 8.3 Theoretical correlation between occupancy q and dissociation cons...
4. Figure 8.4 Case studies illustrating the impact of soaking and co‐crystalliz...
5. Figure 8.5 fHbp epitopes recognized by a monoclonal antibody (12C1) and mapp...
Chapter 9
1. Figure 9.1 Representative structures of membrane proteins (MPs). (a) α‐Helic...
2. Figure 9.2 Workflow from membrane protein production and optimization to cry...
3. Figure 9.3 Statistics of unique integral MP structures and their sources of ...
4. Figure 9.4 Amphipathic molecules used for membrane protein biochemistry. (a)...
5. Figure 9.5 Membrane protein crystallization. (a) Illustration of crystal typ...
Chapter 11
1. Figure 11.1 Lysozyme diffraction data collected for the purpose of experimen...
2. Figure 11.2 Molstack representation of the active site of Pyrococcus yayanos...
3. Figure 11.3 (a) Scatter plot showing structures determined by high‐experienc...
4. Figure 11.4 Distribution of PDB deposits solved by X‐ray crystallography (be...
5. Figure 11.5 R _free as a function of ln(asymmetric unit volume), averaged ov...
Chapter 12
1. Figure 12.1 (a) Comparison between neutron scattering lengths and X‐ray scat...
2. Figure 12.2 (a) The process of crystal growth of ADP‐ribose pyrophosphatase ...
3. Figure 12.3 (a) Bifurcated hydrogen bonding interaction observed in myoglobi...
4. Figure 12.4 Active site structures of HIV‐1 protease. Active site structures...
5. Figure 12.5 Active site structures of serine proteases determined by neutron...
Chapter 13
1. Figure 13.1 A protein 3D model results from a large panel of different NMR m...
2. Figure 13.2 Typical spectra obtained in a 3D triple resonance assignment str...
3. Figure 13.3 Joining assignment and labeling strategies. (a) Full labeling st...
4. Figure 13.4 Timescale of biological events, from the femtosecond to the hour...
5. Figure 13.5 Distance scale of biological objects, from the picometer to the ...
6. Figure 13.6 Molecular motion timescales, energy landscapes, and NMR measurem...
Chapter 14
1. Figure 14.1 NMR and the drug discovery path. Low‐resolution to high‐resoluti...
2. Figure 14.2 Target‐ and ligand‐based NMR experiments.
3. Figure 14.3 NMR ligandability. Outcome of 36 drug discovery projects, binned...
4. Figure 14.4 (a) Two identical spectra are acquired, one without protein targ...
5. Figure 14.5 INPHARMA. One compound binds to the target (“A”) and has magneti...
6. Figure 14.6 Ensemble description of IDPs. (a) Five ensemble states of an IDP...
Chapter 15
1. Figure 15.1 A diagram of methods and data around structural computational ch...
2. Figure 15.2 Human proteome structural coverage based on UniProt–PDB mapping ...
3. Figure 15.3 Sequence identity (TM regions only, as annotated in UniProt) of ...
Chapter 16
1. Figure 16.1 Conserved protein kinases structural core mapped on the catalyti...
2. Figure 16.2 DFG‐in (a) and DFG‐out (b) conformations of the ABL protein kina...
3. Figure 16.3 Chemical structures of the inhibitors discussed in the chapter a...
4. Figure 16.4 (a) Crystal structure of vemurafenib with BRAF ^V600E protein kin...
Chapter 17
1. Figure 17.1 (a) The blood clotting cascade and its regulation. TF, tissue fa...
2. Figure 17.2 Crystal structures of thrombin, FXa and FVIIa. (a) Active site c...
3. Figure 17.3 X‐ray crystal structures of thrombin inhibitor complexes. (a) Me...
4. Figure 17.4 X‐ray crystal structures of FXa complexes with benzamidine‐based...
5. Figure 17.5 (a) Radial distribution of chlorine (middle) and bromine atoms (...
Chapter 18
1. Figure 18.1 Overview of DNA packaging within the cell: DNA is wrapped around...
2. Figure 18.2 Overview of structures of epigenetic proteins (rainbow ribbon). ...
3. Figure 18.3 Select chemical structures of compounds that (a) inhibit deacety...
4. Figure 18.4 Select chemical structures of compounds that (a) inhibit methyla...
Chapter 19
1. Figure 19.1 (a) Ligand binding positions from complex crystal structures for...
2. Figure 19.2 Druggability analysis for a selection of GPCR Class A ligands. (...
3. Figure 19.3 Class B and F GPCRs. (a) Comparison of the secondary structure o...
4. Figure 19.4 A comparison of the binding sites in the active and inactive sta...
5. Figure 19.5 Activation of β₂AR broken down into respective parts thought to ...
6. Figure 19.6 (a) Structures of the top 10 hits identified by virtual screenin...
7. Figure 19.7 SBDD‐optimized antagonist ligands for the A_2A adenosine receptor...
Chapter 20
1. Figure 20.1 MDM2 and MDMX complex and inhibitor structures. (a) MDM2 (protei...
2. Figure 20.2 IAP and BCL complex and inhibitor structures. (a) XIAP‐BIR3 (sur...
3. Figure 20.3 Bromodomain complex and inhibitor structures. (a) I‐BET762 (stic...
4. Figure 20.4 LBMBT3 and WDR5 complex and inhibitor structures. (a) Tri‐propel...
5. Figure 20.5 IL‐2 complex and inhibitor structures. (a) Chemical structures o...
Chapter 21
1. Figure 21.1 Structure of mAb Hz6F4 isotypes. (a) Optimized Hz6F4 IgG4 refere...
2. Figure 21.2 Intact mAb analysis of Hz6F4 isotypes. ESI‐MS mass spectra of Hz...
3. Figure 21.3 Middle‐up analysis of Hz6F4 isotypes. (a) Total ion chromatogram...
4. Figure 21.4 Bottom‐up analysis of Hz6F4 isotypes. (a) Bottom‐up analysis to ...
5. Figure 21.5 IM‐MS analysis of Hz6F4 isotypes in native conditions. Driftscop...
Chapter 22
1. Figure 22.1 HIV‐1 and HCV NS3/4A protease structures. (a) HIV‐1 protease has...
2. Figure 22.2 The conserved binding modes of viral substrates of HIV‐1 and HCV...
3. Figure 22.3 (a) Chemical structure of HIV‐1 protease inhibitors. They are al...
4. Figure 22.4 Guiding drug design using the dynamic substrate envelope. (a) Th...
5. Figure 22.5 A structure‐based drug design scheme that incorporates protein d...
Chapter 23
1. Figure 23.1 Schematic of Mtb targets inhibited by existing drugs or drug can...
2. Figure 23.2 Crystal structure of InhA^S94A complexed with pyridomycin. The ch...
3. Figure 23.3 The RNA polymerase β‐subunit complex. Ribbon representation of t...
4. Figure 23.4 Cartoon representation of the Mtb GyrBA core in complex with DNA...
5. Figure 23.5 Cartoon representation of the M. smegmatis DprE1‐FAD in complex ...
6. Figure 23.6 Cartoon representation of M. phlei c9 ring in complex with BDQ (...
Chapter 24
1. Figure 24.1 A brief history of P450 crystal structures.
2. Figure 24.2 Strategies to lower metabolic clearance include blocking the sit...
3. Figure 24.3 (a) Overlapping of CYP3A4 co‐crystal structures showed flexible ...
4. Figure 24.4 Heat map plot of CYP1A2 (a), CYP2C9 (b), CYP2D6 (c), and CYP3A4 ...
Chapter 25
1. Figure 25.1 Druggable intrinsic disorder‐based interaction between p53 and M...
2. Figure 25.2 Druggable intrinsic disorder‐based interaction between c‐Myc and...
3. Figure 25.3 Fragment probe mapping identifies potential small‐molecule bindi...
4. Figure 25.4 An artificial molecular tweezer for suppression of protein aggre...
Chapter 26
1. Figure 26.1 The cryo‐EM single‐particle analysis (SPA) workflow.
2. Figure 26.2 ProteoPlex. pH optimization of sample behavior guided by Differe...
3. Figure 26.3 The SPA cryo‐EM workflow with EPU. After the grid containing the...

List of Contributors

Ruben Abagyan
Skaggs School of Pharmacy and Pharmaceutical Sciences
University of California
San Diego
La Jolla
CA, USA

Motoyasu Adachi
National Institutes for Quantum and Radiological Science and Technology
Japan Atomic Energy Agency
Tokai, Japan

Mauro Angiolini
Eudendron
Varese
Italy

Daniel Ayoub
Centre d’Immunologie Pierre‐Fabre
Saint‐Julien en Genevois
France

Ivet Bahar
Department of Computational and Systems Biology
School of Medicine
University of Pittsburgh
Pittsburgh
PA, USA

Ahmet Bakan
Department of Computational and Systems Biology
School of Medicine
University of Pittsburgh
Pittsburgh
PA, USA

Shibom Basu
Center of Applied Structural Discovery at Biodesign Institute
School of Molecular Sciences
Arizona State University
Tempe
AZ, USA
and
European Molecular Biology Laboratory
Grenoble Outstation
Grenoble
France

Alain Beck
Centre d’Immunologie Pierre‐Fabre
Saint‐Julien en Genevois
France

Andreas Bender
Department of Chemistry
University of Cambridge
Cambridge
UK

Amandine Boeuf
Centre d’Immunologie Pierre‐Fabre
Saint‐Julien en Genevois
France

P. Ann Boriack‐Sjodin
Epizyme, Inc.
Cambridge
MA, USA
and
Accent Therapeutics, Inc.,
Lexington
USA

Andrea Bortolato
Sosei Heptares
Cambridge
UK

Gérard Bricogne
Global Phasing Limited
Cambridge
UK

Babs Briels
Division of Medicinal Chemistry
Vrije Universiteit Amsterdam
Amsterdam
The Netherlands

Chun‐wa Chung
Medicinal Sciences and Technology
GlaxoSmithKline R&D
Stevenage
UK

Sarah Cianférani
Laboratoire de Spectrométrie de Masse BioOrganique
Institut Pluridisciplinaire Hubert Curien
Université de Strasbourg, CNRS
Strasbourg
France

Ben J. Davis
Vernalis Research
Cambridge
UK

François Debaene
Laboratoire de Spectrométrie de Masse BioOrganique
Institut Pluridisciplinaire Hubert Curien
Université de Strasbourg, CNRS
Strasbourg
France

Sacha De Carlo
DECTRIS Ltd.
Baden‐Dättwil
Switzerland

Francesca Deflorian
Sosei Heptares
Cambridge
UK

Chris de Graaf
Division of Medicinal Chemistry
Vrije Universiteit Amsterdam
Amsterdam
The Netherlands

Carien Dekker
Protein Sciences, Chemical Biology & Therapeutics,
Novartis Institutes for Biomedical Research
Novartis Pharma AG
Basel
Switzerland

Marc‐André Delsuc
Institut de Génétique et de Biologie Moléculaire et Cellulaire
Illkirch
France

Hélène Diemer
Laboratoire de Spectrométrie de Masse BioOrganique
Institut Pluridisciplinaire Hubert Curien
Université de Strasbourg, CNRS
Strasbourg
France

Anindita Dutta
Department of Computational and Systems Biology
School of Medicine
University of Pittsburgh
Pittsburgh
PA, USA

Ursula Egner
Bayer AG
Pharmaceuticals, Research & Development
Berlin
Germany

Anthony Ehkirch
Laboratoire de Spectrométrie de Masse BioOrganique
Institut Pluridisciplinaire Hubert Curien
Université de Strasbourg, CNRS
Strasbourg
France

Petra Fromme
Center of Applied Structural Discovery at Biodesign Institute
School of Molecular Sciences
Arizona State University
Tempe
AZ, USA

Raimund Fromme
Center of Applied Structural Discovery at Biodesign Institute
School of Molecular Sciences
Arizona State University
Tempe
AZ, USA

Arnaud Goepfert
Galapagos SASU
Romainville
France

Marek Grabowski
Department of Molecular Physiology and Biological Physics
University of Virginia
Charlottesville
VA, USA

Michael M. Hann
Medicinal Sciences and Technology
GlaxoSmithKline R&D
Stevenage
UK

Michael Hennig
leadXpro AG
Villigen
Switzerland

Roman C. Hillig
Bayer AG
Pharmaceuticals, Research & Development
Berlin
Germany

Geoffrey A. Holdgate
Hit Discovery, Discovery Sciences
BioPharmaceuticals R&D
AstraZeneca
Alderley Park
UK

Roderick E. Hubbard
Vernalis Research
Cambridge
UK
and
York Structural Biology Laboratory
University of York
York
UK

Andrey V. Ilatovskiy
Skaggs School of Pharmacy and Pharmaceutical Sciences
University of California
San Diego
La Jolla
CA, USA
and
Division of Molecular and Radiation Biophysics
Konstantinov Petersburg Nuclear Physics Institute
NRC Kurchatov Institute
Gatchina
Russia

Bruno Kieffer
Institut de Génétique et de Biologie Moléculaire et Cellulaire
Illkirch
France

Ryota Kuroki
Japan Atomic Energy Agency
Tokai, Japan

Chang Liu
Department of Computational and Systems Biology
School of Medicine
University of Pittsburgh
Pittsburgh
PA, USA

Karolina A. Majorek
Department of Molecular Physiology and Biological Physics
University of Virginia
Charlottesville
VA, USA
and
CRUK Beatson Institute
Glasgow
UK

Jonathan S. Mason
Sosei Heptares
Cambridge
UK

Hans Matter
Sanofi‐Aventis Pharma Deutschland GmbH
Frankfurt am Main
Germany

Wladek Minor
Department of Molecular Physiology and Biological Physics
University of Virginia
Charlottesville
VA, USA

Lionel Mourey
Institut de Pharmacologie et de Biologie Structurale (IPBS)
Université de Toulouse, CNRS, UPS
Toulouse
France

Minh Chau Nguyen
Institut de Pharmacologie et de Biologie Structurale (IPBS)
Université de Toulouse, CNRS, UPS
Toulouse
France

Ayşegül Özen
Department of Biochemistry and Molecular Pharmacology
University of Massachusetts Medical School
Worcester
MA, USA
and Blueprint Medicines
Cambridge
USA

Jean‐Denis Pedelacq
Institut de Pharmacologie et de Biologie Structurale (IPBS)
Université de Toulouse, CNRS, UPS
Toulouse
France

Christopher Phillips
Structure, Biophysics and FBLG, Discovery Sciences, BioPharmaceuticals R&D
AstraZeneca
Cambridge
UK

Hervé‐William Rémigy
ThermoFisher Scientific
Eindhoven
The Netherlands

Jean‐Paul Renaud
Urania Therapeutics
Ostwald
France

Arne Christian Rufer
Lead Discovery, pRED, Pharma Research & Early Development
F. Hoffmann‐La Roche Ltd
Basel
Switzerland

Gebhard F. X. Schertler
Division of Biology and Chemistry‐Laboratory of Biomolecular Research
Paul Scherrer Institute
Villigen
Switzerland
and
Department of Biology
ETH Zürich
Zürich
Switzerland

Celia A. Schiffer
Department of Biochemistry and Molecular Pharmacology
University of Massachusetts Medical School
Worcester
MA, USA

Herman Schreuder
Sanofi‐Aventis Pharma Deutschland GmbH
Frankfurt am Main
Germany

Dennis Scott
Pfizer
Groton
CT, USA

Ivan G. Shabalin
Department of Molecular Physiology and Biological Physics
University of Virginia
Charlottesville
VA, USA

Indira H. Shrivastava
Department of Computational and Systems Biology
School of Medicine
University of Pittsburgh
Pittsburgh
PA, USA

Hao Sun
Pfizer
Groton
CT, USA

Benjamin G. Tehan
Sosei Heptares
Cambridge
UK

Thomas C. Terwilliger
Los Alamos National Laboratory
Los Alamos
NM, USA

Ching‐Ju Tsai
Division of Biology and Chemistry‐Laboratory of Biomolecular Research
Paul Scherrer Institute
Villigen
Switzerland

Vladimir N. Uversky
Department of Molecular Medicine and USF Health
Byrd Alzheimer’s Research Institute
Morsani College of Medicine
University of South Florida
Tampa
FL, USA
and
Laboratory of New Methods in Biology
Institute for Biological Instrumentation
Russian Academy of Sciences
Pushchino
Russia

Alain Van Dorsselaer
Laboratoire de Spectrométrie de Masse BioOrganique
Institut Pluridisciplinaire Hubert Curien
Université de Strasbourg, CNRS
Strasbourg
France

Marc Vitorino
NovAliX
Illkirch
France

Elsa Wagner‐Rousset
Centre d’Immunologie Pierre‐Fabre
Saint‐Julien en Genevois
France

Daniel F. Wyss
Screening, Target and Compound Profiling
Merck Research Laboratories
Kenilworth
NJ, USA

Edward R. Zartler
Quantum Tessera Consulting
Collegeville
PA, USA

Heping Zheng
Department of Molecular Physiology and Biological Physics
University of Virginia
Charlottesville
VA, USA

Matthew D. Zimmerman
Department of Molecular Physiology and Biological Physics
University of Virginia
Charlottesville
VA, USA
and
Commonwealth Computer Research, Inc.
Charlottesville
VA, USA

Preface

Compared to the age of the Earth, the presence of human life on it represents the last four seconds of a day. The same probably holds true for rational drug design compared to the immemorial quest of humans for medicines. Science has been an organized human endeavor since the seventeenth century, and it is only since the second half of the twentieth century that the combined progress of physics, chemistry, biology, and computer science have given birth to molecular structural biology, the basis for rational drug design.

Not only is structural biology a young discipline, but it is also experiencing these days tremendous developments, and thus it is a very exciting time for practitioners involved in its application to drug discovery, where spectacular technical advances are meeting more and more diverse innovative approaches for therapeutic intervention.

On the technological side, we have moved from standard macromolecular X‐ray crystallography at third‐generation synchrotron radiation facilities to burgeoning new developments. Serial femtosecond crystallography at X‐ray free‐electron laser (XFEL) facilities has enabled macromolecular structure determination at room temperature using nanocrystals, yielding physiologically more relevant structures while overcoming both radiation damage and the need for large crystals, which can be game‐changing for challenging targets such as membrane proteins. XFEL also brings new insights into dynamics through time‐resolved studies leading to experimental “molecular movies,” in particular on ligand binding, that will certainly be useful for drug design. Besides, classical X‐ray crystallography has been reinventing itself with serial microcrystallography at synchrotron micro‐focus beamlines, in situ diffraction, automated crystal harvesting, multi‐axis goniometers, etc. NMR is also constantly evolving with innovative methods for both ligand‐observed NMR to monitor target–ligand interactions at high throughput and protein‐observed NMR to solve structures and obtain detailed information on binding sites and on the dynamics of binding, with the help of higher magnetic fields, more sensitive probes, and residue‐specific labeling schemes. But recently, the most spectacular advances have come from the side of single‐particle cryo‐electron microscopy (cryo‐EM): the convergence of many technical improvements in sample preparation, in microscopes, in detectors, and in software has revolutionized the use of this imaging technique, leading to a wealth of high‐resolution structures of large and/or dynamic macromolecular assemblies, including many that could not be crystallized. In particular, single‐particle cryo‐EM will certainly play a major role for the study of membrane protein complexes. Another recently developed cryo‐EM technique, micro‐electron diffraction (MicroED), has been shown to yield high‐resolution structures from well‐ordered nanocrystals, but its wide applicability to macromolecular assemblies needs to be further assessed.

Structure‐based drug design is nowadays more and more relying on dynamic information, both from experiments and from simulations, and on the integration of complementary informations provided by a variety of biophysical techniques, in particular on the thermodynamics of binding, even though it cannot be used in a predictive fashion up to now and needs to be discussed in correlation with very‐high‐resolution structures to be meaningful, and on the kinetics on binding, although the concept of residence time should not be considered alone but in relation with pharmacokinetics. Finally, progress in structural bio‐/chemo‐informatics has been essential to complement experimental techniques with virtual screening and dynamics simulations and for the search of optimized chemical scaffolds now taking into account the flexibility of both the target and the ligand and the energetics of bound water molecules.

On the therapeutic side, the repertoire of drug modalities and targets has widened considerably in the recent years. Besides small molecules and biologics, new territories of drug chemical space are being explored, including hybrids such as antibody–drug conjugates (ADCs), macrocycles, peptides and peptidomimetics, RNA, DNA, genome editing systems, and cells. Targets once thought to be undruggable or highly challenging such as membrane proteins and protein–protein interactions are now actively pursued. For more established targets, new drug candidates are designed to act not only at active sites but also at allosteric sites to achieve higher selectivity. The classical single‐target approach has opened to the multi‐target approach (polypharmacology) to tackle complex diseases. A protein of interest can now be targeted either directly or at the level of its encoding gene (gene editing), its mRNA (gene silencing, exon skipping, etc.), its transcriptional or translational regulation (regulatory factor modulation), or even its degradation (PROTAC™s). In all of these new therapeutic strategies, there is a thrilling game to play for structural biology in the frame of a renewed and extended partnership with medicinal chemistry, for instance, to design specific probes, PROTAC™s, or engineered CRISPR/Cas variants for improved genome editing.

The book gathers contributions from expert practitioners in the field and is divided into four sections:

Part I: Overview, concepts, and approaches.

Part II: Tools, with a strong focus on experimental techniques.

Part III: Case studies of structure‐based discovery on important, established but still challenging therapeutic target families, as well as on monoclonal antibodies as an example of biotherapeutics.

Part IV: Some present‐day frontiers such as intrinsically disordered proteins, neglected diseases, or viral resistance, and promising techniques for the future (XFELs and cryo‐EM) have recently experienced spectacular developments but are not yet ready to be used on a routine basis in drug discovery because of some remaining technical bottlenecks and issues such as throughput, access to high‐end equipments, and availability of experienced practitioners. However, progress in these fields is rapid and should be carefully monitored for drug discovery to benefit as soon as possible from the latest technological advances.

We hope this book will spark interest and communicate enthusiasm for the exciting current developments in the structural biology aspects of drug discovery. In this complex field, it is crucial that structural biologists work hand in hand with medicinal chemists, computational scientists, pharmacologists, and others in order to synergize their respective efforts and boost the development of new drugs. The future holds in the combined use of all available techniques according to the French saying “faire feu de tout bois” to make the best of the expanding structural biology and biophysics toolbox in order to accelerate drug discovery. Early‐stage drug discovery is probably at the dawn of a new era that will see a burst of new biological knowledge and innovative therapeutic approaches. Let us hope these new advances will translate into efficient and safe new therapeutic treatments for mankind.

Because this book encountered a number of difficulties along the way, I would like to wholeheartedly thank my editor Jonathan Rose for his extraordinary patience and my wife Christiane for her support and resilience. And last but not least, I am grateful to the many coauthors – the success of this book will be theirs.

Jean‐Paul Renaud

Structural Biology in Drug Discovery

Methods, Techniques, and Practices

List of Contributors

Preface

Part I
Overview, Concepts, and Approaches