Protein crystallography in drug discovery

The rational, structure-based approach has become standard in present-day drug design. As a consequence, the availability of high-resolution structures of target proteins is more often than not the basis for an entire drug development program. Protein structures suited for rational drug design are almost exclusively derived from crystallographic studies, and drug developers are relying heavily on the power of this method.
Here, researchers from leading pharmaceutical companies present valuable first-hand information, much of it published for the first time. They discuss strategies to derive high-resolution structures for such important target protein classes as kinases or proteases, as well as selected examples of successful protein crystallographic studies. A special section on recent methodological developments, such as for high-throughput crystallography and microcrystallization, is also included.
A valuable companion for crystallographers involved in protein structure determination as well as drug developers pursuing the structure-based approach for use in their daily work.

Author Notes

Robert E. Babine has diverse drug discovery experience over the past 20 years and is presently the Director of Structural & Computational Chemistry at SPRL in Cambridge, Massachusetts. After receiving his Ph.D. in synthetic organic chemistry at Brown University he joined the medicinal chemistry group at Lederle Laboratories. During his 10 years at Lederle his research evolved into structure-based drug design, culminating in a project that discovered hydroxylaminepentanamide HIV protease inhibitors. Thereafter, he was in the medicinal chemistry group at Agouron Pharmaceuticals where he was involved in the early phase discovery of rhinovirus 3C protease inhibitors. After a 2-year stay at Eli Lilly he joined the new startup company SPRL in 2000.
******
Sherin S. Abdel-Meguid is Chief Scientific Officer and Founder of Suntory Pharmaceutical Research Laboratories (SPRL), a structure-based drug discovery company located in Cambridge, Massachusetts. He joined SPRL from SmithKline Beecham (SB) where he was Director of Macromolecular Sciences and Structural Biology, and director of the IL18, Herpes virus protease and CD28 programs. Prior to his work for SB, he was Head of Biophysical Sciences and Protein Engineering at Monsanto. At Monsanto, he built one of the first macromolecular crystallography groups in the pharmaceutical industry. He holds a Ph.D. in Physical Chemistry and M.S. and B.S. degrees in Biochemistry.

Robert E. BabineJerry L. Adams and James Veal and Lisa ShewchukTsunehiro Mizushima and Tomitake TsukiharaJeffrey L. HansenDaniel F. Veber and Maxwell D. CummingsTeruki HonmaSandra M. Waugh and Robert J. FletterickDonald F. Doyle and Lauren J. SchwimmerLei Jin and Robert E. BabineHarren JhotiCarl L. Hansen and Morten Sommer and Kyle Self and James M. Berger and Stephen R. Quake

Preface	p. XI
A Personal Foreword	p. XIII
List of Contributors	p. XV
1 Molecular Recognition of Nuclear Hormone Receptor-Ligand Complexes	p. 1
1.1 Introduction	p. 1
1.1.1 Nuclear Hormone Receptors: Ligand Binding Domains	p. 1
1.1.2 Dimerization and Interactions with Co-activators and Co-repressors	p. 2
1.2 Steroid Receptors	p. 5
1.2.1 The Role of the Ligand	p. 5
1.2.1.1 Estradiol Estrogen Receptor Complex	p. 5
1.2.1.2 Other Estrogen Receptor Agonists Complexes	p. 8
1.2.1.3 Estrogen Receptor Antagonists Complexes	p. 9
1.2.1.4 Genistein - An ER-[beta] Partial Agonist	p. 12
1.2.1.5 R,R-5,11-cis-Diethyl-5,6,11,12-tetrahydrochrysene-2,8-diol: An ER-a Agonist and ER-[beta] Antagonist	p. 14
1.2.2 Structural Basis for Agonism and Antagonism and Partial Agonism: The Role of the Ligand	p. 15
1.2.3 Progesterone/Progesterone Receptor Complex	p. 16
1.2.4 Androgen Receptor Complexes	p. 17
1.2.5 Glucocorticoid Receptor	p. 18
1.2.6 Steroid Ligand Selectivity	p. 19
1.3 The Vitamin D Receptor-Ligand Complexes	p. 22
1.4 The Retinoic Acid Receptors RAR and RXR	p. 23
1.4.1 Introduction	p. 23
1.4.2 RAR-[gamma] and RXR-a Retinoid Complexes	p. 25
1.4.3 Selectivity of RAR Ligands and RAR Isotypes	p. 27
1.4.4 RXR Complexes with Unnatural Ligands	p. 33
1.5 PPAR: Isotype-Selective Ligands	p. 34
1.6 Summary	p. 41
1.7 Acknowledgements	p. 43
1.8 References	p. 43
2 Kinases	p. 47
2.1 Introduction	p. 47
2.2 Structure and Function	p. 48
2.2.1 Tertiary Structure	p. 48
2.2.2 Catalysis and Substrate Binding	p. 49
2.2.3 Regulation and Conformational Flexibility	p. 50
2.2.3.1 Activation Loop Conformation	p. 50
2.2.3.2 Glycine Rich Loop	p. 51
2.2.3.3 C-Helix Orientation	p. 51
2.2.3.4 Lobe Orientation	p. 53
2.2.3.5 Solvent Channel	p. 53
2.3 Crystallization	p. 54
2.3.1 Defining the Construct	p. 54
2.3.2 Mutagenesis	p. 55
2.3.3 Phosphorylation	p. 55
2.4 Inhibitor Design	p. 56
2.4.1 Binding in ATP Cleft	p. 56
2.4.1.1 ATP Binding Sites	p. 57
2.4.1.2 Gatekeeper-Dependent Binding Pocket	p. 60
2.4.1.3 Lipophilic Plug	p. 61
2.4.1.4 Polar Surface Site	p. 61
2.4.2 Conformational Considerations	p. 63
2.4.2.1 Inhibitor-Induced Binding	p. 63
2.4.2.2 What is the Most Appropriate Enzyme Form for Crystallography?	p. 64
2.4.2.3 Homology Models and Surrogate Kinases	p. 66
2.4.3 Paradigms for Kinase Drug Discovery	p. 68
2.4.3.1 High Throughput Screening	p. 68
2.4.3.2 Structure-Based Design	p. 69
2.4.3.3 Mechanism-Based and Ligand Mimetic Design	p. 72
2.4.3.4 Computational Chemistry and Virtual Screening	p. 72
2.4.4 Selectivity	p. 73
2.5 Conclusion	p. 75
2.6 References	p. 76
3 The Proteasome as a Drug Target	p. 83
3.1 Introduction	p. 83
3.2 The Ubiquitin-Proteasome System	p. 83
3.2.1 Role of the Ubiquitin-Proteasome System	p. 84
3.2.2 26S Proteasome	p. 85
3.2.3 20S Proteasome	p. 85
3.3 Structure of the 20S Proteasome	p. 86
3.3.1 Active Sites of Eukaryotic 20S Proteasomes	p. 88
3.3.2 Novel Ntn-Hydrolase Active Site of the [beta]7 Subunit	p. 88
3.3.3 Predicted Structure of Immunoproteasome and Substrate Specificities	p. 89
3.4 Proteasome Inhibitors	p. 93
3.4.1 Structure of Proteasome Inhibitor Complexes	p. 95
3.5 Conclusions	p. 96
3.6 Acknowledgements	p. 96
3.7 References	p. 96
4 Antibiotics and the Ribosome	p. 99
4.1 Introduction	p. 99
4.2 The Ribosome	p. 99
4.2.1 Introduction	p. 99
4.2.2 Binding of tRNA	p. 100
4.2.3 Peptidyl Transferase Activity	p. 101
4.2.4 Structure of the Ribosome	p. 102
4.3 Antibiotics	p. 103
4.3.1 Introduction	p. 103
4.3.2 Antibiotics that Bind to the 50S Subunit	p. 104
4.3.3 MLS[subscript B] Antibiotics	p. 105
4.3.4 Macrolides	p. 107
4.3.4.1 Macrolides, 15- and 16-Membered	p. 107
4.3.4.2 Binding Interactions Between the Lactone Ring and the Ribosome	p. 108
4.3.4.3 Sugar Interactions with the Ribosome	p. 110
4.3.4.4 A Covalent Bond	p. 111
4.3.4.5 Macrolides, 14-Membered	p. 112
4.3.4.6 Rational Drug Design of Macrolides	p. 112
4.3.5 Lincosamides	p. 114
4.3.6 Streptogramins	p. 114
4.3.7 Chloramphenicol	p. 115
4.3.8 Nucleoside Analogue Antibiotics	p. 117
4.3.8.1 Puromycin	p. 117
4.3.8.2 Aminoacyl-4-aminohexosyl-cytosine Antibiotics	p. 118
4.3.9 Other Antibiotics that Bind to the 50S Subunit	p. 119
4.3.9.1 Sparsomycin	p. 119
4.3.9.2 Anisomycin	p. 120
4.4 Prospects for Rational Drug Design of Antibiotics that Bind to the Ribosome	p. 120
4.5 Acknowledgements	p. 122
4.6 References	p. 122
5 Structure-Based Design of Cathepsin K Inhibitors	p. 127
5.1 Introduction	p. 127
5.2 Background and Issues to be Addressed Using Protein Structure	p. 128
5.3 Cysteine Protease Inhibitors: Historical Perspective	p. 131
5.4 Diaminoketone-Based Inhibitors	p. 133
5.5 Cyclic Diaminoketone Inhibitors	p. 136
5.6 Alkoxymethyl and Thiomethyl Dipeptidyl Ketone-Based Inhibitors	p. 140
5.7 Diacylcarbohydrazides	p. 142
5.8 Conclusions	p. 143
5.9 References	p. 145
6 Structure-Based Design of Potent and Selective Cdk4 Inhibitors	p. 147
6.1 Introduction	p. 147
6.2 Homology Modeling of Cdk4	p. 148
6.3 Analysis of the ATP Binding Pocket	p. 151
6.4 Strategies for Structure-Based Lead Identification: Virtual Screening of Known Compounds and de Novo Design	p. 152
6.4.1 New de Novo Design Strategies	p. 153
6.4.2 Evaluation of Chemical Availability by SEEDS	p. 155
6.5 Structure-Based Generation of a New Class of Potent Cdk4 Inhibitors	p. 157
6.5.1 Identification of New Scaffold Candidates Using LEGEND and SEEDS	p. 157
6.5.2 From the Identified Scaffold to Lead Compounds	p. 159
6.5.3 Confirmation of the Binding Mode	p. 159
6.6 Structure-Based Design of Cdk4 Selective Inhibitors	p. 160
6.6.1 Identification of Cdk4-Specific Amino Acid Residues	p. 161
6.6.2 Library Design Based on the Locations of Cdk4-Specific Amino Acid Residues	p. 163
6.7 Conclusion	p. 167
6.8 Acknowledgements	p. 167
6.9 References	p. 168
7 Crystallization and Analysis of Serine Proteases with Ecotin	p. 171
7.1 What is Ecotin?	p. 171
7.2 Methods	p. 173
7.2.1 Expression of Wild Type Ecotin	p. 173
7.2.2 Purification of Wild Type Ecotin	p. 174
7.2.3 Crystallization of Ecotin and Protease Complexes	p. 175
7.3 Representative Examples of Ecotin and Protease Structures	p. 175
7.3.1 Ecotin Defines the S7 Through S4' Subsites of Collagenase	p. 176
7.3.2 Ecotin as a Tight Binding Substrate	p. 177
7.3.3 Ecotin Defines Regions Distal to the Factor Xa Protease Domain	p. 178
7.4 Crystallization and Structure Determination of E[subscript 2]P[subscript 2] Complexes	p. 180
7.5 Conclusion	p. 183
7.6 References	p. 184
8 X-ray Crystallography in the Development of Orthogonal Ligand-Receptor Pairs	p. 187
8.1 Introduction	p. 187
8.2 Applications of OLRPs	p. 188
8.2.1 Applications in Basic Research	p. 188
8.2.2 Applications in Applied Research	p. 189
8.3 Early Work	p. 189
8.4 Structure-Guided Mutagenesis and OLRPs	p. 190
8.4.1 Binding Pocket Mutations	p. 191
8.4.1.1 Nuclear Receptors	p. 191
8.4.1.2 Estrogen Receptor OLRPs	p. 193
8.4.1.3 Retinoic Acid Receptor OLRPs	p. 195
8.4.1.4 Retinoid X Receptor OLRPs	p. 195
8.4.2 Distant Mutations	p. 197
8.5 Other Examples of OLRPs	p. 200
8.5.1 Chemical Inducers of Dimerization to Control Transcription	p. 200
8.5.2 OLRPs and ATP Analogues	p. 202
8.6 Summary	p. 204
8.7 References	p. 206
9 Engineering Proteins to Promote Crystallization	p. 209
9.1 Introduction	p. 209
9.2 Removing Protein Heterogeneity by Truncation	p. 210
9.3 Removing Protein Heterogeneity by Point Mutation	p. 211
9.4 Improving Crystal Packing by Point Mutation	p. 212
9.5 Acknowledgements	p. 214
9.6 References	p. 214
10 High-throughput Crystallography	p. 217
10.1 Introduction	p. 217
10.2 Technological Advances	p. 218
10.2.1 Clone to Crystal	p. 218
10.2.1.1 Protein Production	p. 218
10.2.1.2 Crystallization	p. 219
10.2.2 Crystal to Structure	p. 220
10.2.3 Progress in Structural Genomics	p. 221
10.3 High-throughput Crystallography in Lead Discovery	p. 223
10.3.1 Protein-Ligand Crystal Structures	p. 224
10.4 Fragment-Based Lead Discovery	p. 225
10.4.1 Fragment-Based Lead Discovery Using X-ray Crystallography	p. 226
10.4.2 Structure-Based Optimization of Fragment Hits	p. 229
10.5 Conclusions	p. 230
10.6 Acknowledgements	p. 231
10.7 References	p. 231
11 Micro-Crystallization	p. 235
11.1 Introduction	p. 235
11.2 Microfluidics--Method and Design	p. 237
11.3 Utility of Microfluidics for Crystallization	p. 242
11.4 References	p. 253
Subject Index	p. 257

Available:*

On Order

Summary

Summary

Author Notes

Table of Contents