Structure-Based Drug Discovery

Cover_978-1-61779-519-0......Page 1
FrontMatter......Page 2
Structure-Based Drug Discovery......Page 4
Preface......Page 6
Contents......Page 8
Contributors......Page 10
1. Introduction......Page 12
2. The Information Content of Protein Crystal Structures......Page 13
3. Using Structure in Target Selection and Product Profile Development......Page 24
4.1. Random and Pharmacophore-Based Fragment Screening......Page 26
5. Using X-Ray Crystallography in Lead Optimization......Page 30
6. Summary......Page 36
References......Page 37
1. Introduction......Page 40
2. Construct Design......Page 41
3. Boundaries......Page 42
4. Choosing an Expression System......Page 44
4.1. Prokaryotic Expression Systems......Page 45
4.2.2. Yeast......Page 48
4.2.3. Mammalian Cells......Page 49
5.1. Identifying Regions of Disorder......Page 50
5.2. Reducing Heterogeneity......Page 52
5.4. Post-translational Modifications......Page 53
6. Conclusion......Page 55
References......Page 56
1. Introduction......Page 60
1.1. Expression of Proteins in Escherichia coli......Page 61
1.2. Cell Lysis and Pre-chromatography Processing......Page 62
1.3. Immobilized Metal Affinity Chromotography......Page 63
1.4.1. SDS-PAGE......Page 64
1.5. Ion Exchange Chromatography......Page 65
1.6. Size-Exclusion Chromatography......Page 66
1.8. The Purification and Crystallization of Human Hsp90......Page 68
2.1. Cell Culture and Lysis......Page 69
2.6. Protein Concentration and Crystallization......Page 70
3.3. Gravity Flow Ion Exchange Chromatography......Page 71
3.6. Size-Exclusion Chromatography (Gel Filtration)......Page 72
3.8.1. Protein Concentration......Page 73
4. Notes......Page 74
References......Page 75
1. Introduction......Page 78
1.1. Background......Page 79
2. Materials......Page 83
2.1. Plates and Accessories......Page 85
3. Methods......Page 86
3.1. Initial Screening......Page 87
3.2. Cryoprotection and Diffraction Screening......Page 89
3.3. Optimization Methods......Page 93
3.4. Alternative Optimization Methods......Page 98
4. Summary......Page 100
References......Page 101
1. Introduction......Page 104
2. X-Rays......Page 105
2.1.1. Physics of X-Rays......Page 106
2.1.2. X-Ray Generators......Page 108
2.1.3. Synchrotrons......Page 110
Bending Magnets......Page 112
Insertion Devices (Wigglers and Undulators)......Page 114
2.1.4. Future Sources......Page 115
2.2. X-Ray Optics......Page 117
2.2.1. Mirrors......Page 118
Crystal Monochromators......Page 119
2.3. Beamline......Page 122
Overview......Page 123
Fast Shutter, Collimator, Slits, Beamstop, Intensity Monitor......Page 124
Goniometer and Crystal Positioning......Page 125
Sample Cooling......Page 126
Sample Mounter......Page 127
X-Ray Area Detector (CCD)......Page 128
3.1. Preparations......Page 131
3.2. Sample Tracking......Page 132
3.3.2. Evaluation of Screening Images......Page 133
3.3.3. Crystal Screening at Takeda San Diego......Page 136
3.4. Data Collection Strategies......Page 138
Detector-to-Sample Distance, 2- Q Angle, and Beam Divergence......Page 139
Exposure Time......Page 140
Starting Spindle Axis Rotation Angle, Rotation Angle Range, and Oscillation Angle......Page 142
Beam Size and Detector Binning......Page 143
3.5.2. Indexing and Refinement......Page 144
3.5.3. Indexing Problems......Page 145
3.5.4. Integration and Scaling......Page 146
3.5.5. Finding the Correct Space Group......Page 147
3.6.3. Beamline “Etiquette”......Page 148
4. Outlook......Page 149
References......Page 150
1. Introduction......Page 154
2. Requirements for Molecular Graphics Applications in Drug Discovery......Page 155
3. Requirements for X-Ray Structures for Drug Design......Page 156
4. Protein–Ligand Complex Modelling Using Coot......Page 157
4.2. Terminal Residue Addition......Page 158
4.5. Dictionary Generation......Page 159
4.6.3. MakeTNT......Page 160
4.8.2. PURY......Page 161
4.9. Ligand Fitting......Page 162
4.10. Conformation Generation......Page 163
5.2. Electrostatic Surface Representation......Page 164
5.4. Assessment of Ligand Geometry......Page 165
6. Views and Annotations......Page 166
7. The Future of Coot......Page 167
References......Page 169
1. Introduction......Page 172
2.1. Compound Suppliers......Page 175
2.4. Software for Protein Crystal Structure Analysis......Page 176
3.1.2. Compound Properties and the Rule of Three......Page 177
3.1.4. Creating a Compound List......Page 178
3.2.1. Factors Determining the Number of Compounds in a Mixture Group......Page 180
3.2.2. Creating Shape Diverse Mixtures......Page 181
3.3.1. Data Collection Facilities and Robotics......Page 182
3.3.2. Crystal Requirements for Successful Fragment Binding......Page 183
3.4.1. High-Throughput Structure Solution......Page 184
4. Notes......Page 186
References......Page 188
1. Introduction......Page 190
2. Selection of Enzymes for Assay......Page 191
3.1. ATPase Assays......Page 196
Materials......Page 197
Materials......Page 198
3.2.1. Isothermal Titration Calorimetry......Page 199
3.2.2. Fluorescence Polarization......Page 201
3.3. DNA Topology Assays......Page 202
3.3.1. Agarose Gel Electrophoresis......Page 203
3.3.2. DNA Triplex Formation......Page 204
4.1.1. Malachite Green......Page 205
4.1.2. Enzchek Assay......Page 206
4.2.1. Isothermal Titration Calorimetry......Page 208
4.2.2. Fluorescence Polarization......Page 209
4.3.1. Agarose Gel Electrophoresis......Page 210
4.3.2. DNA Triplex Formation......Page 212
5. Notes......Page 213
References......Page 216
1. Introduction......Page 220
2.1. Background......Page 221
2.2. Structural Features of the Type IIA Topoisomerases......Page 222
2.3. Identification of the “Hot Spots” in the GyrB Binding Cavity......Page 225
2.4.1. Structure-Based Virtual Screening......Page 226
Target Preparation......Page 227
Fundamentals of Molecular Docking......Page 228
Flexible Receptor Docking......Page 230
2.4.2. Identification of New Lead Compounds by Virtual Screening......Page 232
2.5. Structure-Based Lead Optimization......Page 235
3. Concluding Remarks......Page 239
References......Page 240
1.1. Shape as a Concept......Page 246
1.3. Computationally Designed Compound Libraries......Page 247
1.4. The Concept of Similarity......Page 248
2.1.3. OEChem Toolkit (TK)......Page 249
3.1.1. Hard-Sphere Models......Page 250
3.2.2. Core and Reagent Selection......Page 251
3.2.6. Use of Protein Shape as a Positive/Negative Filter......Page 252
3.2.7. Diversity Libraries......Page 253
3.3. Shape and Electrostatics, in the Context of Bioisosterism......Page 254
4.1.4. Shape Is Not Equally Important at All Positions of a Molecule......Page 258
5. Summary......Page 259
References......Page 260
1. Introduction......Page 262
2. Identification of Water Molecules in Protein–Ligand Complexes......Page 264
3. Water and the Thermodynamics of Ligand Binding......Page 266
References......Page 276
1. Introduction......Page 278
2.1. Molecular Biology......Page 280
2.4. Purification of FLAP......Page 281
2.5. Crystallization of FLAP......Page 282
2.6. Data Collection and Processing......Page 283
2.7. Low-Resolution Structure Determination and Refinement......Page 284
3.1. Description of the FLAP Structure......Page 286
3.2. Inhibitor Binding Site......Page 290
3.3. Modelling Compounds into the Inhibitor-Binding Site......Page 293
3.4. Central Pocket of FLAP......Page 295
3.5. Proposed Mechanism of Arachidonic Acid Transfer......Page 296
References......Page 298
1. Introduction......Page 302
2. Dihydrofolate Reductase (DHFR): Addressing the Issue of Resistance to the First-Generation Drug......Page 304
3. Dihydro-neopterin Aldolase (DHNA): Bad Target or Bad Drug?\r......Page 312
4. Peptide Deformylase (PDF): Different Strategies for Peptidomimics......Page 314
5. Fatty Acid Synthesis (FabI): Narrow Spectrum by Design (or Accident)......Page 321
6. Lesson Learned......Page 325
7. Summary......Page 327
References......Page 328
1. Introduction......Page 332
2. Antibody Structure......Page 333
2.3. The Fab......Page 335
2.4. The Antigen Combining Site......Page 337
2.6. The Hinge......Page 338
4.1. Humanization Approaches......Page 339
4.2. CDR Grafting......Page 340
4.3. Framework Selection......Page 341
4.4. FR Libraries......Page 342
4.5. Engineering CDR’s for Enhanced Human Content......Page 343
4.6. Affinity Optimization......Page 344
5.1. Nature’s Fcs: IgG Isotypes......Page 345
5.2. Optimized Fcs......Page 346
5.3. Fc Engineering for Extended Half-Life......Page 348
6.2. Chemical Modifications......Page 349
6.3. Physical Stability......Page 351
6.4. Aggregation......Page 352
7. Conclusions/Future Directions......Page 354
References......Page 355
1. Introduction......Page 362
3. Considerations for Structure-Based Design: Optimizing Drug-Like Properties......Page 363
4. Physicochemical Parameters in Optimization of Drug Candidates......Page 364
4.1.1. Structure and Design of Orally Acting Factor Xa Inhibitors......Page 365
4.2. Comparing Selectivity Modes in Isoforms of Nitric Oxide Synthase: Design of Selective nNOS Inhibitors......Page 371
4.3. Isoform Specific Structural Plasticity in NOS: Anchored Plasticity Approach to iNOS Selective Inhibitors......Page 379
5. Crystal Packing, Pharmaceutical Co-crystals and Physicochemical Properties; Drug Solubility and Absorption......Page 383
6. Concluding Remarks......Page 388
References......Page 389
BackMatter_INDEX......Page 394