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XXII Contents
12.10 Expression Patterns on aChip: Microarray Technology ................................ 182
12.11 SNPs and Polymorphism: What Makes Us Dierent
12.12 The Personal Genome: Access to an Individualized Therapy?
12.13 When Genetic Dierences Turn into Disease
.................................... 183
.......................... 184
........................................... 184
12.14 Epigenetics: Lifestyle and Environment Inuence Gene Activity Like aPen
Leaves aMark in the Book of Life
12.15 The Scope and Limitations of Gene Therapy
12.16 Synopsis
................................................................................ 189
Bibliography and Further Reading
Experimental Methods of Structure Determination ............................ 193
13
13.1 Crystals: Aesthetic on the Outside, Periodic on the Inside
13.2 Just Like Wallpaper: Symmetries Govern Crystal Packings
13.3 Crystal Lattices Diract X-Rays
....................................................... 185
........................................... 187
....................................................... 190
............................. 194
............................. 196
......................................................... 196
13.4 Crystal Structure Analysis: Evaluating the Spatial Arrangement and Intensity of
Diraction Patterns
13.5 Diraction Power and Resolution Determine the Accuracy of aCrystal Structure
13.6 Electron Microscopy: Topographic Images Reveal Macromolecular Structures
13.7 Structures in Solution: The Resonance Experiment in NMR Spectroscopy
13.8 From Spectra to Structure: Distance Maps Evolve into Spatial Geometries
13.9 How Relevant Are Structures in aCrystal or NMR Tube to aBiological System?
13.10 Synopsis
................................................................................ 212
Bibliography and Further Reading
..................................................................... 197
..... 201
........ 205
............. 208
............ 209
........ 211
....................................................... 213
Three-Dimensional Structure of Biomolecules .................................. 215
14
14.1 The Amide Bond: Backbone of Proteins
14.2 Proteins Fold in Space to Form
α
................................................ 216
-Helices and β-Strands ............................... 217
14.3 From Secondary Structure Via Motifs and Domains to Tertiary and
Quaternary Structure
14.4 Are the Fold Structure and Biological Function of Proteins Correlated?
14.5 Proteases Recognize and Cleave Substrates in Well-Tailored Pockets
14.6 From Substrate to Inhibitor: Screening of Substrate Libraries
................................................................... 220
............... 223
.................. 224
......................... 224
14.7 When Crystal Structures Learn to Move: From Static Structures to Dynamics and
Reactivity
............................................................................... 226
14.8 Solutions to the Same Problem: Serine Proteases with Diering Folds
Have Identical Function
14.9 DNA as aTarget Structure of Drugs
14.10 Synopsis
................................................................................ 230
Bibliography and further reading
Molecular Modeling ................................................................. 233
15
15.1 3D Structural Models as Well-Established Tools in Chemistry
15.2 Strategies in Molecular Modeling
15.3 Knowledge-Based Approaches
15.4 Force Field Methods
15.5 Quantum Chemical Methods
15.6 Computing and Analyzing Molecular Properties
15.7 Molecular Dynamics: Simulation of Molecular Motion
15.8 Dynamics of aFlexible Protein in Water
15.9 Model and Simulation: Where Are the Dierences?
15.10 Synopsis
................................................................................ 244
Bibliography and further reading
................................................................ 227
..................................................... 228
........................................................ 231
.......................... 234
...................................................... 234
......................................................... 235
.................................................................... 236
........................................................... 237
....................................... 239
................................. 239
................................................ 242
.................................... 243
........................................................ 245
Contents
XXIII
16 Conformational Analysis ........................................................... 247
16.1 Many Rotatable Bonds Create Large Conformational Multiplicity
16.2 Conformations Are the Local Energy Minima of aMolecule
16.3 How to Scan Conformational Space Eciently?
........................................ 249
16.4 Is It Necessary to Search the Entire Conformational Space?
The Diculty in Finding Local Minima Corresponding to the Receptor-Bound State
16.5
..................... 248
............................ 249
............................ 250
... 251
16.6 An Eective Search for Relevant Conformations by Using aKnowledge-Based
Approach
16.7 What Is the Outcome of aConformational Search?
16.8 Synopsis Bibliography and Further Reading
Quantitative Structure-Activity Relationships
IV
............................................................................... 252
..................................... 253
................................................................................ 253
....................................................... 253
and Design Approaches
17 Pharmacophore Hypotheses and Molecular Comparisons .................... 257
17.1 The Pharmacophore Anchors aDrug Molecule in the Binding Pocket
17.2 Structural Superposition of Drug Molecules
17.3 Logical Operations with Molecular Volumes
........................................... 258
........................................... 259
17.4 The Pharmacophore is Modied by Conformational Transitions
17.5 Systematic Conformational Search and Pharmacophore Hypothesis:
The “Active Analog Approach”
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 262
17.6 Molecular Recognition Properties and the Similarity of Molecules
17.7 Automated Molecular Comparisons and Superpositioning Based on Recognition
Properties
17.8 Rigid Analogues Trace the Biologically Active Conformation
.............................................................................. 264
.......................... 266
17.9 If Rigid Analogues are Lacking: Model Compounds Elucidate the Active
Conformation The Protein Denes the Pharmacophore: “Hot Spot” Analysis of the Binding Pocket
17.10
........................................................................... 266
17.11 Searching for Pharmacophore Patterns in Databases Generates Ideas for Novel
Lead Compounds
17.12 Synopsis
................................................................................ 271
Bibliography and Further Reading
....................................................................... 270
....................................................... 272
................. 258
....................... 260
.................... 263
... 267
Quantitative Structure–Activity Relationships. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 273
18
18.1 How It All Began: Structure–Activity Relationships of Alkaloids
18.2 From Richet, Meyer, and Overton to Hammett and Hansch
18.3 The Determination and Calculation of Lipophilicity
18.4 Lipophilicity and Biological Activity
.................................................... 275
18.5 The Hansch Analysis and the Free–Wilson Model
.................................... 275
...................................... 276
18.6 Structure–Activity Relationships of Molecules in Space
18.7 Structural Alignment as aPrerequisite for the Relative Comparison of Molecules
18.8 Binding Anities as Compound Properties
18.9 How Is aCoMFA Analysis Performed?
18.10 Molecular Fields as Criteria of aComparative Analysis
............................................ 278
.................................................. 279
................................. 280
18.11 3D-QSAR: Correlation of Molecular Fields with Biological Properties
....................... 274
............................ 274
............................... 278
..... 278
.................. 280
18.12 Results of aComparative Molecular Field Analysis and Their Graphical Interpretation
18.13 Scope, Limitations, and Possible Expansions of the CoMFA Analysis
................... 283
18.14 A Glimpse Behind the Scenes: Comparative Molecular Field Analysis of Carbonic
Anhydrase Inhibitors
18.15 Synopsis
................................................................................ 287
Bibliography and Further Reading
................................................................... 284
....................................................... 288
282
XXIV Contents
19
From In Vitro to In Vivo: Optimization of ADME and Toxicology Properties
19.1 Rate Constants of Compound Transport
19.2 Absorption of Organic Molecules: Model and Experimental Data
19.3 The Role of Hydrogen Bonds
........................................................... 294
19.4 Distribution Equilibria of Acids and Bases
Absorption Proles of Acids and Bases ................................................... 296
19.5
............................................... 292
...................... 294
.............................................. 295
.. 291
19.6 What Is the Optimal Lipophilicity of aDrug? ........................................... 298
19.7 Computer Models and Rules to Predict ADME Parameters
19.8 From In Vitro to In Vivo Activity
........................................................ 300
19.9 Compartmentalization: Natural Ligands Are Often Unspecic
19.10 Specicity and Selectivity of Drug Interactions
19.11 Of Mice and Men: The Value of Animal Models
19.12 Toxicity and Adverse Eects
............................................................ 304
19.13 Animal Protection and Alternative Test Models
19.14 Synopsis Bibliography and Further Reading
Protein Modeling and Structure-Based Drug Design .......................... 309
20
................................................................................ 306
....................................................... 308
........................................ 301
......................................... 302
........................................ 306
20.1 Pioneering Studies in Structure-Based Drug Design
20.2 Strategies in Structure-Based Drug Design
............................................ 311
20.3 Search Tools for Databases of Experimentally Determined Protein Complexes
20.4 Comparison of Protein-Binding Pockets
................................................ 312
20.5 High Sequence Identity Facilitates Model Generation
............................. 299
......................... 300
................................... 310
........ 312
................................. 312
20.6 Secondary Structure Prediction and Amino Acid Replacement Propensities Support
Model Building at Low Sequence Identity
20.7 Ligand Design: Seeding, Expanding, and Linking
20.8 Docking Ligands into Binding Pockets
20.9 Scoring Functions: Ranking of Constructed Binding Geometries
20.10 De Novo Design: From LUDI to the Automated Assembly of Novel Ligands
20.11 The Feasibility of Designing Ligands In Silico
20.12 Synopsis Bibliography and Further Reading
................................................................................ 320
....................................................... 320
.............................................. 314
...................................... 316
................................................. 316
...................... 318
............ 318
.......................................... 319
A Case Study: Structure-Based Inhibitor Design for tRNA-Guanine
21
Transglycosylase
21.1 Shigellosis: Disease and Therapeutic Options
21.2 Blocking Pathogenesis on the Molecular Level
21.3 The Crystal Structure of tRNA-Guanine Transglycosylase as aStarting Point
21.4 A Functional Assay to Determine Binding Constants
21.5 LUDI Discovers the First Leads
21.6 Surprise: AFlipped Amide Bond and aWater Molecule
..................................................................... 323
.......................................... 325
......................................... 325
.......... 326
................................... 326
.......................................................... 329
................................ 330
21.7 Hot Spot Analysis and Virtual Screening Open the Floodgate to New Ideas
for Synthesis
21.8 The Filling of Hydrophobic Pockets and Interference with aWater Network
21.9 With aSalt Bridge: Finally Nanomolar!
21.10 Surprise: The Enzyme is Only Functional as aDimer
21.11 Site-directed Mutagenesis: What Binds the Dimer Together
21.12 When Nothing Else Works: Chemical Poking at the Contact Interface
21.13 Only Serendipity Can Help: Dierent Crystal Form—New Dimer
21.14 Tracking the Dynamic Transformation with the Appropriate Spins
............................................................................ 331
........... 332
................................................. 334
................................... 338
........................... 340
. . . . . . . . . . . . . . . . . . 342
...................... 343
.................... 343
21.15 When Sulfur Accidentally Oxidizes and Starts aFragment Design Project in aNew
Arrangement
........................................................................... 346
21.16 A Fragment Opens aTransient Pocket and Suggests the Design of Bacteria-specic
Inhibitors
21.17 Many Ways to aSmart Antibiotic Against Shigellosis
21.18 Synopsis Bibliography and Original Papers
............................................................................... 349
.................................. 350
................................................................................ 352
........................................................ 353
Contents
XXV
V Drugs and Drug Action: Sucesses of Structure-Based Design
22 How Drugs Act: Concepts for Therapy ............................................ 357
22.1 The Druggable Genome
22.2 Enzymes as Catalysts in Cellular Metabolism
22.3 How Do Enzymes Push Substrates Towards the Transition State?
22.4 Enzymes and Their Inhibitors
22.5 Receptors as Target Structures for Drugs
22.6 Drugs Regulate Ion Channels: Our Extremely Fast Switches
22.7 Blocking Transporters and Water Channels
22.8 Modes of Action: ANever-Ending Story
22.9 Resistance and Its Origin
22.10 Combined Administration of Drugs
22.11 Synopsis
................................................................................ 368
Bibliography and Further Reading
Inhibitors of Hydrolases with an Acyl–Enzyme Intermediate ................. 371
23
23.1 Serine-Dependent Hydrolases
23.2 Structure and Function of Serine Proteases
23.3 The S
Pocket of Serine Proteases Determines Specicity .............................. 374
1
23.4 Seeking Small-Molecule Thrombin Inhibitors
23.5 Design of Orally Available Low Molecular Weight Elastase Inhibitors
23.6 Serine Protease Inhibitors: Thrombin Was Just the Starting Point
23.7 Serine, aFavored Nucleophile in Degrading Enzymes
23.8 Triads in All Variations: Threonine as aNucleophile
23.9 Cysteine Proteases: Sulfur, the Big Brother of Oxygen as aNucleophile in the Triad
23.10 Synopsis
................................................................................ 400
Bibliography and Further Reading
................................................................ 358
........................................... 359
...................... 360
........................................................... 361
............................................... 362
........................... 364
............................................ 364
................................................ 365
............................................................... 367
.................................................... 368
....................................................... 369
......................................................... 372
............................................ 372
.......................................... 376
.................. 384
..................... 385
................................. 390
.................................... 394
... 396
....................................................... 400
Aspartic Protease Inhibitors ....................................................... 403
24
24.1 Structure and Function of Aspartic Proteases
24.2 Design of Renin Inhibitors
.............................................................. 405
24.3 Design of Substrate Analogue HIV Protease Inhibitors
24.4 Structure-Based Design of Nonpeptidic HIV Protease Inhibitors
24.5 The Development of Resistance Against HIV Protease Inhibitors
24.6 A Basic Nitrogen as aPartner for the Aspartic Acids of the Catalytic Dyad
24.7 Other Targets from the Family of Aspartic Proteases
24.8 Synopsis Bibliography and Further Reading
Inhibitors of Hydrolyzing Metalloenzymes ...................................... 427
25
25.1 Structure of Zinc Metalloproteases
................................................................................ 423
....................................................... 424
..................................................... 428
25.2 Key Step in the Design of Metalloprotease Inhibitors: Binding to the Zinc Ion
25.3 Thermolysin: Tailored Design of Enzyme Inhibitors
25.4 Captopril, aMetalloprotease Inhibitor for Hypertension Therapy
25.5 Finally the Crystal Structure of ACE: Does aSuccess Story Have to Be Rewritten?
.......................................... 404
................................. 411
....................... 413
...................... 416
............. 418
................................... 423
........ 429
.................................... 431
..................... 432
..... 434
25.6 Inhibitors of Matrix Metalloproteases: An Approach to Treat Cancer and
Rheumatoid Arthritis?
25.7 Carbonic Anhydrases: Catalysts of aSimple but Essential Reaction A Case for Two: Zinc and Magnesium in the Catalytic Centers of Phosphodiesterases
25.8
25.9 What Zinc Can Do, Iron Can Too
.................................................................. 436
.................... 440
.. 444
........................................................ 446
25.10 Acetyl Group Cleavage Condenses Chromatin and Regulates Reading of Gene
Segments: An Opportunity for Therapy?
25.11 Synopsis
................................................................................ 449
Bibliography and Further Reading
............................................... 447
....................................................... 450
XXVI Contents
26 Transferase Inhibitors ............................................................... 451
26.1 The Kinase “Gold Rush”
26.2 Structure of Protein Kinases: More than 500 Variations with Similar Geometry
26.3 Isosteric with ATP, and Selective Nonetheless?
®
26.4 Gleevec
: Success Stories Breed Copycats! ............................................. 458
26.5 Tracing Selectivity: The Bump-and-Hole Method
26.6 Metals Teach Kinase Inhibitors Selectivity
26.7 Phosphatases: Reversal Switch to Activate and Inactivate Proteins
26.8 Inhibitors of PTP-1B: Treatment for Diabetes and Obesity?
26.9 Molecular Glue Inhibits the Release of Phosphatase Activity
26.10 Inhibitors of Catechol-O-Methyltransferase
26.11 Blocking the Transfer of Farnesyl and Geranyl Anchors
26.12 Synopsis
................................................................................ 480
Bibliography and Further Reading
Oxidoreductase Inhibitors ......................................................... 483
27
27.1 Redox Reactions in Biological Systems Use Cofactors
27.2 Chemotherapeutics for Cancer and Bacteria: Dihydrofolate Reductase Inhibitors
27.3 HMG-CoA Reductase Inhibitors: The Changing Fate of Drug Development
27.4 Hitting aMoving Target: Aldose Reductase Inhibitors
β
27.5 11
-Hydroxysteroid Dehydrogenase ................................................... 500
27.6 The Cytochrome P450 Enzyme Family
27.7 What Makes Slow and Fast Metabolizers Dierent?
27.8 Blocking the Degradation of Neurotransmitters: Monoamine Oxidase Inhibitors
27.9 Cyclooxygenase: AKey Enzyme in Pain Sensation
27.10 Synopsis
................................................................................ 518
Bibliography and Further Reading
................................................................. 452
. . . . . . . . 453
......................................... 454
...................................... 462
.............................................. 464
.................... 466
............................ 468
.......................... 472
............................................ 473
................................ 477
....................................................... 481
.................................. 484
..... 487
............ 490
................................. 496
.................................................. 502
.................................... 506
..... 508
..................................... 512
....................................................... 519
Agonists and Antagonists of Nuclear Receptors ................................ 521
28
28.1 Nuclear Receptors Are Transcription Factors
28.2 The Structure of Nuclear Receptors
.................................................... 523
28.3 Steroid Hormones: How Small Dierences Translate to the Receptor
28.4 Helix Open, Helix Closed: How Agonists and Antagonists Are Dierentiated
28.5 Agonists and Antagonists of Steroid Hormone Receptors
28.6 Ligands of PPAR Receptors
............................................................. 531
28.7 Ligands of Nuclear Receptors Stimulate Metabolism
28.8 Synopsis Bibliography and Further Reading
Agonists and Antagonists of Membrane-Bound Receptors ................... 537
29
................................................................................ 535
....................................................... 536
29.1 The Family of G-Protein-Coupled Receptors
29.2 Rhodopsins Provide the First Models of G-Protein-Coupled Receptors
β
29.3 Structure of the Human
-Adrenergic Receptor ....................................... 541
2
........................................... 522
.................. 523
.......... 525
............................. 527
.................................. 533
............................................ 538
................ 540
29.4 How Does aGPCR Communicate with Its Macromolecular Protein Partners
in the Cell?
29.5 Peptide-Binding Receptors: Development of AngiotensinII Antagonists
.............................................................................. 544
.............. 547
29.6 Do Peptidic Agonists and Small-Molecule Antagonists Bind at the Same Position
of the AT
29.7 Lessons Taught by the Nose: We Smell with GPCRs
Receptor? .................................................................... 548
1
..................................... 551
29.8 Receptor Tyrosine Kinases and Cytokine Receptors: Where Insulin, EPO,
and Cytokines Display Their Activity
29.9 Synopsis
................................................................................ 557
Bibliography and Further Reading
................................................... 552
....................................................... 559
Contents
XXVII
30 Ligands for Channels, Pores, and Transporters ................................. 561
30.1 Electric Potential and Ion Gradients Stimulate Cells
30.2 Molecular Function of aPotassium Channel at the Atomic Level
30.3 Binding Undesirable: The hERG Potassium Channel as an Antitarget
.................................... 562
...................... 564
.................. 567
30.4 Electromechanical Control of Voltage-Dependent Ion Channels : How Small Ligands
Tighten of Loosen a Hydrophobic Belt in Ion Channels
30.5 Tiny Ligands Gate Giant Ion Channels
.................................................. 574
30.6 Ligands Gate as Agonists and Antagonists: The Function of an Ion Channel
30.7 Power Brake Boosters for GABA-Gated Chloride Channels
30.8 The Mode of Action of aVoltage-Gated Chloride Channel
30.9 ATP Hydrolysis Fuels Ion Flux Against Concentration Gradients
30.10 Transporters: The Gatekeepers to the Cell
.............................................. 587
30.11 Membrane Passage in Bacteria: Pores, Carriers, and Channel Formers
30.12 Aquaporins Regulate the Cellular Water Inventory
30.13 Synopsis Bibliography and Further Reading
Ligands for Surface Receptors ..................................................... 597
31
31.1 The Family of Integrin Receptors
................................................................................ 592
....................................................... 594
....................................................... 598
31.2 Successful Design of Peptidomimetic Fibrinogen Receptor Antagonists
31.3 Selectins: Surface Receptors Recognizing Carbohydrates
31.4 Fusion Inhibitors Impede Viral Invasion
................................................ 605
31.5 Neuraminidase Inhibitors Prevent Budding of Mature Viruses
31.6 Stopping the Common Cold: Inhibitors for the Capsid Protein of Rhinovirus
31.7 MHC Molecules: Where the Immune System Presents Peptide Fragments
31.8 Synopsis Bibliography and Further Reading
................................................................................ 622
....................................................... 623
................................ 569
........... 576
............................. 579
............................. 585
....................... 586
................. 590
..................................... 591
.............. 600
.............................. 603
......................... 607
.......... 612
............. 616
Biologicals: Peptides, Proteins, Nucleotides, and Macrolides as Drugs ..... 625
32
32.1 Gene-Technological Production of Proteins
32.2 Tailored Modications to Insulin
Monoclonal Antibodies as Vaccines, Chemotherapeutics, and Receptor Antagonists
32.3
....................................................... 627
32.4 Antisense Oligonucleotides and mRNA as Drugs?
32.5 Nucleosides and Nucleotides as False Substrates
32.6 Molecular Wedges Destroy Protein–Nucleotide Recognition
............................................ 626
.. 628
..................................... 633
...................................... 636
.......................... 639
32.7 Macrolides: Microbial Warheads as Potential Cytostatics, Antimycotics,
Immunosuppressants, or Antibiotics
32.8 Synopsis
................................................................................ 651
Bibliography and Further Reading
................................................... 643
....................................................... 652
Service Part
Illustration Source References ........................................................... 656
Name Index Subject Index
............................................................................. 663
........................................................................... 667

About the author

Gerhard Klebe
is Professor of Medicinal Chemistry at Philipps University in Marburg, Germany. He retired in April 2020. He has been teaching pharmaceutical and medicinal chemistry, drug action and drug design for almost 30 years. His research has focused on structure-activity relationships, 3D-QSAR methods, conformational and pharmacophore analysis, docking methods, database analysis, protein crystallography, structure-based drug design, and biophysical characterization of protein-ligand interactions (https://agklebe.pharmazie.uni-marburg.de/?id=&lang=en). The thermodynamic characterization and the involvement of water molecules in the binding process have been a particular focus. A number of well known and worldwide applied computer tools have been developed in his laboratory (CoMSIA, AFMoC, ReLiBase, Cavbase, Drugscore, Mobile). With his research group, he has worked on a signicant number of the drug targets also covered in the book. In total, his group has contributed more than 1600 crystal structures to the publicly available PDB. Together with colleagues at the Bessy synchrotron in Berlin, his group estab­lished a dedicated beamline for fragment-based lead discovery, including many tools for further hit-to-lead optimization. Out of his research group came the start-up company CrystalsFirst (www.crystalsrst.com), which focuses on crystallographic fragment screening using sophisti- cated protein crystal stabilization techniques and combining crystal screening with computational design. Prior to his appointment in Marburg, he taught drug design and crystallography at the University of Heidelberg and worked for more than a decade in an industrial setting in drug discovery at BASF AG in Ludwigshafen, Germany.
Foundations
in Drug Research
I
This colored copper engraving from the probably most beautiful plant book, the Hortus Eystettensis von Basilius Besler, Eichstätt, 1613, shows the sea onion, Scilla alba (today: Urginea maritimaL.). It was already known to the ancient Egyptians, Greeks, and Romans as aremedy for many illnesses, e.g., dropsy (today: congestive heart failure). It was also worshipped as ageneral repellent against misfortune. It was not until the 20th century that the cardioactive glycosides scillaren and proscillavidin contained in the sea onion were isolated in pure form and introduced into therapy
®
as such or in the form of the more bioavailable derivative meproscillarin (Clift
).
Contents
Chapter 1 Drug Research: Yesterday, Today, and Tomorrow – 3
Chapter 2 In the Beginning, There Was Serendipity – 15
Chapter 3 Classical Drug Research – 23
Chapter 4 Protein–Ligand Interactions as the
Basis for Drug Action – 39
Chapter 5 Optical Activity and Biological Eect – 67
Drug Research: Yesterday,
Today, and Tomorrow
Contents
1.1 It All Began with Traditional Medicines – 5
1.2 Animal Experiments as aStarting Point for Drug Research – 6
1.3 The Battle Against Infectious Disease – 7
1.4 Biological Concepts in Drug Research – 7
1.5 In Vitro Models and Molecular Test Systems – 8
1.6 The Successful Therapy of Psychiatric Illness – 9
1.7 Modeling and Computer-Aided Design – 10
1.8 The Results of Drug Research and the Drug Market – 11
1.9 A Subject of Conict: Pharmaceuticals – 12
1.10 Synopsis – 13
Bibliography and Further Reading – 14
© The Author(s), under exclusive license to Springer-Verlag GmbH, DE, part of Springer Nature 2024 G. Klebe, Drug Design, https://doi.org/10.1007/978-3-662-68998-1_1