Molecular Biology in Medicinal Chemistry

Contents Preface xv Foreword xvii Contributors xix Part I Molecular Targets 1 1 Cellular Assays in Drug Discovery 3 Hugo Albrecht, Daniela Brodbeck-Hummel, Michael Hoever, Beatrice Nickel and Urs Regenass 1.1 Introduction 3 1.1.1 Positioning Cellular Assays 3 1.1.2 Impact on Drug Discovery 4 1.1.3 Classification of Cellular Assays 5 1.1.4 Progress in Tools and Technologies for Cellular Compound Profiling 7 1.2 Membrane Proteins and Fast Cellular Responses 8 1.2.1 Receptors 8 1.2.1.1 FLIPR Technology for Detection of Intracellular Calcium Release 9 1.2.1.2 Competitive Immunoassay for Detection of Intracellular cAMP 9 1.2.1.3 Enzyme Fragment Complementation (EFC) Technology 12 1.2.2 Membrane Transport Proteins 12 1.2.2.1 Ion Channels 13 1.2.2.2 MDR Proteins 16 1.3 Gene and Protein Expression Profiling in High-throughput Formats 17 1.3.1 Reporter Gene Assays in Lead Finding 17 1.3.2 Reporter Gene Assays in Lead Optimization 21 1.4 Spatio-temporal Assays and Subpopulation Analysis 24 1.4.1 Phosphorylation Stage-specific Antibodies 25 1.4.2 Target-protein-specific Antibodies 26 1.4.3 Protein–GFP Fusions 27 1.4.4 Fluorescence Resonance Energy Transfer (FRET) 29 1.4.5 GPCR Activation using Bioluminescence Resonance Energy Transfer (BRET) 31 v 1.4.6 Protein Fragment Complementation Assays (PCA) 31 1.5 Phenotypic Assays 33 1.5.1 Proliferation/Respiration/Toxicity 33 1.5.2 Apoptosis 34 1.5.3 Differentiation 35 1.5.4 Monitoring Cell Metabolism 36 1.5.5 Other Phenotypic Assays 38 Acknowledgments 39 References 39 2 Gene Knockout Models 48 Peter Ruth and Matthias Sausbier 2.1 Introduction 48 2.2 Gene Knockout Mice 48 2.2.1 ES Cells 49 2.2.2 Targeting Vector 52 2.2.3 Selection of Recombinant ES Cells 54 2.2.4 Injection of Recombinant ES Cells into Blastocysts and Blastocyst Transfer to Pseudopregnant Recipients 54 2.2.5 Chimeras and F1 and F2 Offspring 56 2.3 Tissue-Specific Gene Expression 59 2.3.1 Ligand-Activated CRE Recombinases 61 2.3.2 The Tetracycline/Doxycycline-Inducible Expression System 63 2.4 Transgenic Mice 65 2.5 Targeted Gene Disruption in Drosophila 67 2.6 Targeted Gene Knockdown in Zebrafish 68 2.7 Targeted Caenorhabditis Elegans Deletion Strains 70 References 71 3 Reporter Gene Assay Systems for the Investigation of G-protein-coupled Receptors 73 Michaela C. Dinger and Annette G. Beck-Sickinger 3.1 Receptors and Cellular Communication 73 3.1.1 Ion Channel-linked Receptors 73 3.1.2 Enzyme-linked Cell-surface Receptors 74 3.1.3 GPCRs 75 3.2 Affinity and Activity of GPCR Ligands 77 3.3 The Role of Transcription Factors in Gene Expression 78 3.3.1 CREB 79 3.3.2 SRF 79 3.3.3 STAT Proteins 79 3.3.4 c-Jun 80 3.3.5 NF-AT 80 3.4 Reporter Genes 80 3.4.1 CAT 81 vi Contents 3.4.2 b-Gal 81 3.4.3 b-Glucuronidase 82 3.4.4 AP 83 3.4.5 SEAP 83 3.4.6 b-Lactamase 83 3.4.7 Luciferase 84 3.4.8 GFP 85 3.5 Reporter Gene Assay Systems for the Investigation of GPCRs 85 3.5.1 Application of Luciferase as a Reporter Gene 86 3.5.2 Application of other Reporter Genes for the Investigation of GPCRs 88 References 91 4 From the Human Genome to New Drugs: The Potential of Orphan G-proteincoupled Receptors 95 Remko A. Bakker and Rob Leurs 4.1 Introduction 95 4.2 GPCRs and the Human Genome 97 4.2.1 GPCR Architecture, Signaling and Drug Action 97 4.2.2 Identification of GPCRs 98 4.2.3 GPCRs in the Postgenomic Era: Orphan Receptors 99 4.3 Ligand Hunting 100 4.3.1 Reverse Pharmacology Approaches to oGPCRs 100 4.3.2 In Silico Approaches 101 4.3.3 Tissue Expression 102 4.3.4 Expression of an oGPCR of Interest 103 4.3.5 Screening Approaches 105 4.4 Screening for oGPCR Ligands using Functional Assays 106 4.4.1 GTPgS-binding Assays 106 4.4.2 Measurements of cAMP 108 4.4.3 Ca2þ Measurements 109 4.4.4 The Cytosensor Microphysiometer 110 4.4.5 Reporter Gene Assays 111 4.4.6 GPCR–G-protein Coupling 111 4.4.7 Ligand-independent GPCR Activity 113 4.4.8 Novel Screening Strategies 114 4.5 Future Prospects 115 References 117 Part II Synthesis 123 5 Stereoselective Synthesis with the Help of Recombinant Enzymes 125 Nagaraj N. Rao and Andreas Liese 5.1 Stereoselective Synthesis before the Advent of Genetic Engineering 125 5.2 Classical Methods of Strain Improvement for Stereoselective Synthesis 126 Contents vii 5.3 Genetic Engineering and the Advent of Recombinant Enzymes for Stereoselective Synthesis 129 5.4 b-Lactam Antibiotics 132 5.4.1 Penicillins 132 5.4.2 Cephalosporins 134 5.5 Polyketide Antibiotics 137 5.6 Vitamins 140 5.6.1 L-Ascorbic Acid (Vitamin C) 140 5.6.2 Biotin (Vitamin B8, Vitamin H) 142 5.6.3 Riboflavin (Vitamin B2) 143 5.6.4 Nicotinamide (Vitamin B3 or PP) 143 5.6.5 Vitamin B12 144 5.6.6 D-Pantothenic Acid (Vitamin B5) 144 5.6.7 Vitamin A 145 5.6.8 Vitamin K (Menaquinone) 146 5.7 Steroids 146 5.8 Other Drugs 146 5.8.1 L-Dihydroxyphenyl Alanine (L-DOPA) 146 5.8.2 Crixivan2 147 5.8.3 N-Acetyl Neuraminic Acid (NANA) 147 5.8.4 Cholesterin Biosynthesis Inhibitors (HMG-CoA Reductase Inhibitors) 148 5.8.5 Omapatrilat 149 5.8.6 Hydromorphone 149 5.9 Concluding Remarks 150 References 150 6 Nucleic Acid Drugs 153 Joachim W. Engels and Jo¨rg Parsch 6.1 Introduction 153 6.2 Chemical Synthesis of Oligonucleotides 153 6.3 Chemical Modifications of Oligonucleotides 155 6.3.1 20-Modifications 156 6.3.2 Alkyl- and Arylphosphonates 157 6.3.3 Phosphorothioates 159 6.3.4 N30-P50-phosphoramidates 160 6.3.5 Morpholino Oligonucleotides 160 6.3.6 PNAs 160 6.3.7 Sterically LNAs 161 6.3.8 Oligonucleotide Conjugates 162 6.3.8.1 50-End Conjugates 162 6.3.8.2 30-End Conjugates 162 6.4 Mechanism of Action 163 6.4.1 The Antisense Concept 163 6.4.1.1 Steric Blocking 163 viii Contents 6.4.1.2 RNase H Activation 165 6.4.2 The Triplex Concept 165 6.4.3 Ribozymes 167 6.4.3.1 Structure and Reaction Mechanism 168 6.4.3.2 Triplet Specificity 169 6.4.3.3 Stabilization of Ribozymes 170 6.4.3.4 Inhibition of Gene Expression 170 6.4.3.5 Colocalization 171 6.4.4 RNAi 171 6.5 Positioning Identification of Ribozyme Accessible Sites on Target RNA 172 6.6 Delivery 173 6.7 Application 175 6.8 Conclusion 175 References 176 Part III Analysis 179 7 Recent Trends in Enantioseparation of Chiral Drugs 181 Bezhan Chankvetadze 7.1 Introduction 181 7.2 Current Status in the Development and use of Chiral Drugs 181 7.3 The Role of Separation Techniques in Chiral Drug Development, Investigation and Use 183 7.4 Preparation of Enantiomerically Pure Drugs 184 7.4.1 Resolution of Racemates 185 7.4.2 Chiral Pool 186 7.4.3 Catalytic Asymmetric Synthesis 187 7.4.4 Chromatographic Techniques 189 7.4.4.1 SMB 189 7.4.4.2 Other Chromatographic and Electrophoretic Techniques for the Preparation of Enantiomers 193 7.5 Bioanalysis of Chiral Drugs 194 7.5.1 GC 195 7.5.2 HPLC 197 7.5.3 CE 198 7.5.4 CEC 203 7.6 Future Trends 204 References 205 8 Affinity Chromatography 211 Gerhard K. E. Scriba 8.1 Introduction 211 8.2 Principles of Affinity Chromatography 212 8.3 The Ligand 214 Contents ix 8.3.1 Monospecific Ligands 215 8.3.2 Group-specific Ligands 215 8.3.3 Ligand Development 217 8.4 The Affinity Matrix 218 8.4.1 The Support Material 218 8.4.2 Immobilization of the Ligand 219 8.5 Principles of Operation 222 8.6 Modes of Affinity Chromatography 223 8.6.1 Bioaffinity Chromatography 223 8.6.2 Immunoaffinity Chromatography 224 8.6.3 Dye-ligand-affinity Chromatography 225 8.6.4 Immobilized Metal Ion-affinity Chromatography (IMAC) 225 8.6.5 Hydrophobic Interaction Chromatography (HIC) 226 8.6.6 Covalent Chromatography 228 8.6.7 Membrane Affinity Chromatography 229 8.7 Applications of Affinity Interactions 229 8.7.1 Purification of Pharmaceutical Proteins 230 8.7.1.1 Plasma Proteins 231 8.7.1.2 Recombinant Proteins 231 8.7.2 Analytical Applications 233 8.7.2.1 HPAC 234 8.7.2.2 Affinity Capillary Electrophoresis 236 8.7.2.3 Affinity Biosensors 237 References 239 9 Nuclear Magnetic Resonance-based Drug Discovery 242 Ulrich L. Gu¨nther, Christina Fischer and Heinz Ru¨terjans 9.1 Introduction 242 9.2 SAR by NMR 243 9.2.1 Sample Preparation 247 9.2.2 Quantitative Evaluation 247 9.2.2.1 Binding Affinities 247 9.2.2.2 Structure Determination from Chemical Shift Perturbations 250 9.2.3 Advanced Technologies in SAR by NMR 250 9.2.4 Data Evaluation 251 9.3 Monitoring Small Molecules 252 9.3.1 Relaxation Methods 253 9.3.2 Diffusion Methods 254 9.3.3 Methods Involving Magnetization Transfer 256 9.3.3.1 Transferred NOE (TrNOE) 256 9.3.3.2 Saturation Transfer 257 9.3.3.3 NOE Pumping 260 9.3.3.4 WaterLOGSY and ePHOGSY 261 9.3.4 Sample Preparation 262 x Contents 9.4 Conclusions 264 Acknowledgments 264 References 265 10 13C- and 15N-Isotopic Labeling of Proteins 269 Christian Klammt, Frank Bernhard and Heinz Ru¨terjans 10.1 Introduction 269 10.2 Expression Systems for the In Vivo Incorporation of 13C and 15N Labels into Proteins 270 10.2.1 Protein Production and 13C and 15N-labeling in E. coli Expression Systems 271 10.2.2 13C- and 15N-labeling of Proteins in P. pastoris 274 10.2.3 13C- and 15N-labeling of Proteins by Expression in Chinese Hamster Ovary (CHO) Cells 276 10.2.4 13C- and 15N-labeling of Proteins in Other Organisms 276 10.2.5 Strategies for the Production of Selectively 13C- and 15N-labeled Proteins 277 10.2.5.1 Selective Labeling of Amino Acids 277 10.2.5.2 Specific Isotope Labeling with 13C 278 10.2.5.3 Segmental Isotope Labeling 279 10.3 Cell-free Isotope Labeling 280 10.3.1 Components of Cell-free Expression Systems 281 10.3.2 Cell-free Expression Techniques 285 10.3.3 Specific Applications of the Cell-free Labeling Technique 289 References 292 11 Application of Antibody Fragments as Crystallization Enhancers 300 Carola Hunte and Cornelia Mu¨nke 11.1 Introduction 300 11.2 Crystallization of Membrane Proteins Mediated by Antibody Fragments 303 11.2.1 Overview 303 11.2.2 Generation of Antibodies Suitable for Co-crystallization 306 11.2.2.1 Immunization 306 11.2.2.2 Selection of Antibodies 307 11.2.2.3 Antibody Fragments 308 11.2.3 Protein Purification and Crystallization 314 11.2.3.1 Indirect Immunoaffinity Purification 314 11.2.3.2 Crystallization 316 11.2.3.3 Alternative Approaches for Expansion of the Polar Surface 317 11.3 Conclusions 317 Acknowledgments 318 References 318 Contents xi Part IV Kinetics, Metabolism and Toxicology 323 12 Pharmacogenetics: The Effect of Inherited Genetic Variation on Drug Disposition and Drug Response 325 Kurt Kesseler 12.1 General 325 12.2 Relationships between Genotype and Phenotype 326 12.3 Establishing Relations between Genotype and Phenotype 327 12.4 Phenotyping versus Genotyping – Prediction of Individual Drug Response 329 12.5 Genetic Polymorphism in DMEs 330 12.6 Polymorphism of CYP2D6 332 12.7 Genetic Polymorphism in Drug Transporters 338 12.8 Multidrug Resistance Gene – Marker versus Functional Polymorphism 338 12.9 Genetic Polymorphisms in Drug Targets 339 12.9.1 Cholesterol Ester Transfer Protein (CETP) Polymorphism as a Biomarker for Response to Lipid-lowering Therapy 340 12.9.2 b2-Adrenergic Receptor: Predictability of Haplotype versus Single SNPs 340 12.9.3 Polygenic Drug Target Polymorphism: Response to Clozapine Treatment 341 12.10 Concluding Remarks 341 References 344 13 Pharmacogenomics of Bioavailability and Elimination 363 Ingolf Cascorbi and Heyo K. Kroemer 13.1 Introduction 363 13.2 Contribution of Metabolism to Drug Clearance 363 13.2.1 CYPs 363 13.2.1.1 CYP2C9 365 13.2.1.2 CYP2C19 366 13.2.1.3 CYP2D6 366 13.2.1.4 CYP3A 369 13.2.2 Phase II Enzymes 369 13.2.2.1 Arylamine N-acetyltransferases (NAT2) 369 13.2.2.2 Thiopurine-S-methyltransferase (TPMT) 370 13.3 Drug Transporters: P-glycoprotein (P-gp) 371 13.4 Conclusion 373 References 373 14 Toxicogenomics: Integration of New Molecular Biological Tools in Toxicology 381 Wilbert H. M. Heijne, Ben van Ommen and Rob H. Stierum 14.1 Developments in Toxicology 381 xii Contents 14.2 Functional Genomics: New Molecular Biological Tools 383 14.2.1 Genomics 383 14.2.2 Transcriptomics 383 14.2.3 Proteomics 384 14.2.4 Metabolomics 386 14.2.5 Data Processing and Bioinformatics 389 14.2.6 Biological Interpretation 390 14.3 Integration of Functional Genomics Technologies in Toxicology 392 14.3.1 Mechanism Elucidation 392 14.3.2 Transcriptomics Fingerprinting and Prediction of Toxicity 393 14.3.3 Identification of Early Markers of Toxicity 393 14.3.4 Interspecies Extrapolation 394 14.3.5 Extrapolation from In Vitro Experiments to the In Vivo Situation 394 14.3.6 Reduction of Use of Laboratory Animals 394 14.3.7 Combinatorial Toxicology 394 14.4 Illustration: The Application of Toxicogenomics to Study the Mechanism of Bromobenzene-induced Hepatotoxicity 395 14.5 Conclusion 397 References 397 Index 399 Contents